Nuclease Resistant Polynucleotides and Uses Thereof

ABSTRACT

The invention provides, among other things, methods of stabilizing mRNA and nuclease resistant mRNA prepared in accordance with such methods. hi certain embodiments, the nuclease resistant mRNA encodes a functional protein, such as enzyme, and is characterized by its resistance to nuclease digestion, increased half-life and/or its ability to produce increased amounts of the functional protein (e.g., enzyme) encoded thereby.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/983,635, filed on May 18, 2018, which is a continuation of Ser. No.14/406,424, filed on Dec. 8, 2014, which is the U.S. National StageEntry claiming priority and benefit to International ApplicationPCT/US2013/044769, filed on Jun. 7, 2013, which claims priority andbenefit to U.S. Provisional Application No. 61/657,465, filed on Jun. 8,2012, the disclosures of each of which are incorporated herein byreference in their entirety for all purposes.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format. Said ASCII copy, created on Sep. 12,2022, is named MRT_1089US3_SL.txt and is 2,277 bytes in size. The entirecontents of the Sequence Listing are herein incorporated by referencefor all purposes.

BACKGROUND

The administration of exogenous nucleic acids and polynucleotides, forexample DNA vectors and plasmids, to a subject for the treatment ofprotein or enzyme deficiencies represents a significant advance in thetreatment of such deficiencies however, the administration of suchexogenous nucleic acids to a subject remains especially challenging. Forexample, gene therapies that rely on viruses to carry and deliverexogenous polynucleotides (e.g., DNA) to host cells and that cause theintegration of such polynucleotides into the host cells' genome arecapable of eliciting serious immunological and inflammatory responses.Furthermore, in certain instances the integration of such exogenouspolynucleotides into the host cells' genome has the potential ofmisregulating the expression of the host's endogenous genes andunpredictably impacting cellular activity.

Similarly, plasmid vector expression systems have represented anattractive alternative approach for gene therapy because of their easeof preparation, stability, and relative safety compared to viralvectors. Such plasmids are however, frequently characterized as havinghighly inefficient cellular uptake in vivo.

To date, the treatment of protein (e.g., enzyme) deficiencies haveprimarily involved the administration of recombinantly-prepared proteins(e.g., enzymes) to the affected subject. While in some instances, theuse of recombinant proteins and enzymes may provide a means ofameliorating the symptoms of the underlying deficiency, the utility ofsuch therapies are often limited and are not considered curative.Furthermore, recombinant proteins or enzymes are often prepared usingnon-human cell lines and may lack certain post-translationalmodifications (e.g., human glycosylation) relative to their endogenouslyproduced counterparts. Such differences may contribute to the lowerefficacy of such recombinantly-prepared proteins or enzymes and/or maycontribute to their immunogenicity and the incidence of adversereactions (e.g., infusion-related reactions such as fever, pruritus,edema, hives and other allergic-like symptoms). Recombinant protein andenzyme replacement therapies are also associated with great financialexpense. For example, the average cost for enzyme replacement therapy inthe United States may approach 5200,000-5300,000 USD per year dependingon the subject's weight and prescribed dose. (Brady, R O., Annual Reviewof Medicine (2006), 57: 283-296.) Since replacement therapies are notcurative, the costs associated with, for example, enzyme replacementtherapies impose a significant burden on the already taxed healthcaresystem. Further contributing to the costs associated with suchtherapies, such therapies often require the administration of multipleweekly or monthly doses, with each such dose being administered by aqualified healthcare professional.

The administration of polynucleotides such as RNA (e.g., mRNA) that donot have to be transcribed may also represent a suitable alternative toprotein or enzyme replacement therapies. While the development ofexogenous therapeutic mRNA polynucleotides encoding functional proteinsor enzymes represents a promising advancement, in practice the utilityof such treatments may be limited by the poor stability of suchpolynucleotides in vivo. In particular, the poor stability of exogenouspolynucleotides may result in the inefficient expression (e.g.,translation) of such polynucleotides, further resulting in a poorproduction of the protein or enzyme encoded thereby. Especiallydetrimental to the ability of mRNA polynucleotides to be efficientlytranslated into a functional protein or enzyme is the environment towhich such polynucleotides are exposed in vivo. Following theadministration of a polynucleotide, the polynucleotide may undergodegradation, for example upon exposure to one or more nucleases in vivo.Ribonucleases endoribonucleases and exoribonucleases) represent a classof nuclease enzymes that are capable of catalyzing the degradation ofRNA polynucleotides into smaller components and thereby render thepolynucleotide ineffective. Nuclease enzymes (e.g., RNase) are thereforecapable of shortening the circulatory half-life (t_(1/2)) of, forexample, exogenous or recombinantly-prepared mRNA polynucleotides. As aresult, the polynucleotide is not translated, the polynucleotide isprevented from exerting an intended therapeutic benefit and its efficacysignificantly reduced.

Previous efforts to stabilize polynucleotides have focused on complexingthe polynucleotide with, for example, a liposomal delivery vehicle.While such means may positively impact the stability of the encapsulatedpolynucleotides, many lipids used as a component of such liposomalvehicles (e.g., cationic lipids) may be associated with toxicity. Otherefforts have been directed towards the modification of one or morenucleotides that comprise the polynucleotide.

Novel, cost effective and therapeutically efficient approaches andtherapies are still needed for the treatment of protein and enzymedeficiencies. Particularly needed are strategies and therapies whichovercome the challenges and limitations associated with theadministration of exogenous mRNA polynucleotides, including for example,novel methods and compositions relating to the stabilization ofexogenous polynucleotides. Also needed are polynucleotides (e.g., RNA)and compositions that exhibit enhanced stability (e.g., increasedhalf-life in vivo) and nuclease resistance and which facilitate theefficient expression or production of functional proteins or enzymes.The development of such stable and/or nuclease resistant compositionsare necessary to overcome the limitations of conventional gene therapyand could provide viable treatments or even cures for diseasesassociated with the aberrant production of proteins or enzymes.

SUMMARY OF THE INVENTION

Disclosed herein are nuclease resistant polynucleotides and relatedcompositions and methods. Such polynucleotides and compositionsgenerally encode functional polypeptides, proteins and/or enzymes (e.g.,an mRNA polynucleotide may encode a functional urea cycle enzyme). Incertain embodiments, such compositions are characterized as being moreresistant to nuclease degradation relative to their unmodified or nativecounterparts.

Disclosed herein are methods of stabilizing or modulating (e.g.,increasing or otherwise improving) the nuclease resistance of apolynucleotide (e.g., an RNA polynucleotide). The polynucleotides thatare the subject of the present inventions preferably encode a functionalexpression product (e.g., a protein or enzyme) and may be generallycharacterized as comprising both a coding region and a non-codingregion. In some embodiments, the methods disclosed herein generallycomprise a step of contacting the non-coding region of thepolynucleotide (e.g., the poly-A tail of an mRNA polynucleotide) with acomplementary (e.g., a perfectly complementary) stabilizingoligonucleotide under suitable conditions, thereby causing thestabilizing oligonucleotide to hybridize to the non-coding region of thepolynucleotide. Upon hybridizing of the stabilizing oligonucleotide(e.g., a 15-mer poly-U oligonucleotide) to the polynucleotide, thepolynucleotide is rendered more resistant to nuclease degradation. Forexample, in certain embodiments, provided herein are methods ofincreasing the nuclease resistance of an mRNA polynucleotide comprisinga poly-A tail by contacting the poly-A tail of such polynucleotide witha complementary poly-U stabilizing oligonucleotide. Upon hybridizing tothe non-coding region of the polynucleotide (e.g., the poly-A tail) toform a duplexed or double-stranded region, nuclease degradation of thepolynucleotide may be reduced, delayed or otherwise prevented. Withoutwishing to be bound by any particular theories, it is believed that theobserved stability and nuclease resistance of the polynucleotidesdisclosed herein is due in part to the single-stranded specificity ofsome nuclease enzymes (e.g., ribonucleases).

In certain embodiments, the stabilizing oligonucleotides disclosedherein may hybridize to the non-coding region of the polynucleotide(e.g., the 5′ or 3′ non-coding regions of an mRNA polynucleotide) so asnot to interfere with the message encoded by the coding region of suchpolynucleotide. Stabilizing oligonucleotides may be prepared such thatthey are perfectly complementary to a fragment of the non-coding region(e.g., perfectly complementary to a fragment of the poly-A tail of anmRNA polynucleotide). For example, the stabilizing oligonucleotide maybe complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 87.5%, 90%, 92.5%, 95%, 97%, 98%, 99% or 100% complementary)to one or more non-coding regions of the polynucleotide selected fromthe group of regions consisting of the 3′ untranslated region (UTR), the5′ untranslated region (UTR), the poly-A tail and a terminal cap.Similarly, the stabilizing oligonucleotide may be complementary (e.g.,perfectly complementary) to a region spanning discreet structures withinthe non-coding region. For example, a stabilizing oligonucleotide may beprepared such that it is perfectly complementary to a region (orfragment of a region) that spans either the 3′ UTR and the poly-A tailor alternatively the 5′ UTR and a 5′ cap structure.

While certain embodiments described herein contemplate the hybridizationof the stabilizing oligonucleotide to the non-coding region of thepolynucleotide, the present inventions are not limited to suchembodiments. Rather, also contemplated are methods and compositions inwhich the stabilizing oligonucleotide hybridizes to a region spanning orcomprising both a fragment of the coding region as well as a fragment ofthe non-coding region of the polynucleotide. In such embodiments(particularly where the non-coding region comprises the poly-A tail ofthe polynucleotide) hybridization to a region of the polynucleotidecomprising fragments of both the coding and non-coding regions mayprovide a means to direct the hybridization of the stabilizingoligonucleotide to a specific region of the polynucleotide.

Also contemplated by the present invention is the administration ofexogenous stabilizing oligonucleotides to a subject, for example, totreat a disease or condition associated with the aberrant expression orunder-expression or production of a protein or enzyme. The foregoing maybe particularly suitable for the treatment of diseases or conditionscharacterized as having a suboptimal or sub-therapeutic endogenousproduction of a protein or enzyme. In such embodiments, an exogenousstabilizing oligonucleotide that is complementary (e.g., perfectlycomplementary) to a region of the under expressed endogenouspolynucleotide (e.g., one or more of the 5′ and/or 3′ UTR) isadministered to a subject. Following administration of the exogenousoligonucleotide, such oligonucleotide may hybridize to the one or moreendogenous polynucleotides (e.g., mRNA) encoding an under-expressedpolypeptide, protein or enzyme such that the stability (e.g., thenuclease resistance) of the endogenous polynucleotide is modulated(e.g., enhanced or otherwise increased). The stabilized or nucleaseresistant endogenous polynucleotide (e.g., mRNA) may be characterized ashaving an increased circulatory half-life (t_(1/2)) and/or an increasedtranslational efficiency relative to its native counterpart, generallycausing the amount of the expression product (e.g., a lysosomal enzyme)encoded by such endogenous polynucleotide to be enhanced or otherwiseincreased. In certain embodiments, the stabilizing oligonucleotide isdelivered or administered in a suitable pharmaceutical carrier orcomposition (e.g., encapsulated in a lipid nanoparticle vehicle).

In some embodiments, the present invention is directed to stable ornuclease resistant polynucleotides (e.g., mRNA) and methods of theirpreparation. Such polynucleotides (e.g., recombinantly-prepared mRNA)may be prepared by hybridizing one or more complementary (e.g.,perfectly complementary) stabilizing oligonucleotides to the codingand/or non-coding regions of the polynucleotide. The polynucleotidesdisclosed herein may encode a functional polypeptide, protein or enzyme.For example, the polynucleotide (e.g., mRNA) may encode a protein orenzyme selected from the group consisting of erythropoietin, humangrowth hormone, cystic fibrosis transmembrane conductance regulator(CFTR), alpha-galactosidase A, alpha-L-iduronidase,iduronate-2-sulfatase, N-acetylglucosamine-1-phosphate transferase,N-acetylglucosaminidase, alpha-glucosaminide acetyltransferase,N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine-4-sulfatase,beta-glucosidase, galactose-6-sulfate sulfatase, beta-galactosidase,beta-glucuronidase, glucocerebrosidase, heparan sulfamidase,hyaluronidase, galactocerebrosidase, ornithine transcarbamylase (OTC),carbamoyl-phosphate synthetase 1 (CPS1), argininosuccinate synthetase(ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1).

Also disclosed herein are methods of treating one or more diseases orconditions associated with a protein or enzyme deficiency or theaberrant expression of one or more nucleic acids. Such methods comprisea step of administering a composition (e.g., a liposomal vehicle)comprising one or more of the nuclease resistant polynucleotides (e.g.,mRNA) of the present invention to a subject affected by such disease orcondition. Following the administration of such compositions to asubject, one or more targeted host cells are transfected and thecontents of such composition delivered intracellularly where it may betranslated and the expression product (e.g., a polypeptide, protein orenzyme) produced. In certain instances, the expression product (e.g., atranslated protein or enzyme) may be excreted extracellularly by the oneor more targeted host cells (e.g., hepatocytes).

Also disclosed herein are stabilized or nuclease resistantpolynucleotides (e.g., mRNA) that comprise a complementary stabilizingoligonucleotide hybridized to the coding and/or non-coding regions ofsuch polynucleotide. In certain embodiments, the stabilizingoligonucleotide and/or the polynucleotide (e.g., mRNA) comprise at leastone modification. The modification of one or both of the polynucleotide(e.g., mRNA) and/or the stabilizing oligonucleotide to incorporate oneor more modifications may be used as a means of further modulating(e.g., enhancing or increasing) the nuclease resistance of thepolynucleotide. Without wishing to be bound by a particular theory, itis believed that the incorporation of modifications (e.g., 2′-O-alkylsugar modifications) to either the stabilizing oligonucleotide and/orthe polynucleotide act to sterically block or delay nuclease degradationof the polynucleotide and thereby improve stability. Accordingly, incertain embodiments, the polynucleotide and/or the stabilizingoligonucleotide (e.g., a poly-U oligonucleotide) comprise at least one(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen or more) modified nucleobase.

Contemplated modifications may include, for example, sugar modificationsor substitutions (e.g., one or more of a 2′-O-alkyl modification, alocked polynucleotide (LNA) or a peptide polynucleotide (PNA).) Inembodiments where the sugar modification is a 2′-O-alkyl modification,such modification may include, but are not limited to a2′-deoxy-2′-fluoro modification, a 2′-O-methyl modification, a2′-O-methoxyethyl modification and a 2′-deoxy modification. In certainembodiments where the modification is a nucleobase modification, suchmodification may be selected from the group consisting of a 5-methylcytidine, pseudouridine, 2-thio uridine, 5-methylcytosine, isocytosine,pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine,2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurinecytosine, and combinations thereof.

In certain embodiments, the contemplated modification may involve theinter-nucleosidic bonds that comprise the stabilizing oligonucleotideand/or the polynucleotides. For example, contemplated modificationsintroduced to one or both of the stabilizing oligonucleotide and/or thepolynucleotide may include one or more phosphorothioate bonds. In oneembodiment, all of the inter-nucleosidic bonds of one or both of thestabilizing oligonucleotide and the polynucleotide are phosphorothioatebonds.

The nuclease resistance of the polynucleotides disclosed herein may becharacterized relative to the native or unmodified counterpartpolynucleotides (e.g., relative to an un-hybridized polynucleotide thathas not been contacted or treated with a stabilizing oligonucleotide).For example, the nuclease resistant polynucleotides disclosed herein maybe at least about two, three, four, five, six, seven, eight, nine, ten,twelve, fifteen, twenty, twenty-five, thirty, fifty, one hundred timesmore stable in vivo relative to their native or un-hybridizedcounterparts. In certain embodiments, the circulatory half-life(t_(1/2)) of the polynucleotide in vivo is indicative of suchpolynucleotide's stability. In other embodiments, the relative amount ofexpression product (e.g., a polypeptide, protein or enzyme) expressed(e.g., translated) from the polynucleotide is indicative of itsstability.

In some embodiments, the present invention relates to methods ofincreasing the quantity of an expression product (e.g., a functionalprotein or enzyme) that is or may be expressed (e.g., translated) from apolynucleotide transcript. For example, such methods may generallycomprise a step of contacting a portion of the coding and/or non-codingregions of an mRNA polynucleotide transcript with a stabilizingoligonucleotide such that the stabilizing oligonucleotide hybridizes tothe mRNA transcript. In certain embodiments, the stabilizingoligonucleotide and the mRNA polynucleotide transcript are contacted atabout a 0.1:1 ratio. In other embodiments, the stabilizingoligonucleotide and the mRNA polynucleotide transcript are contacted atabout a 0.25:1 ratio. In yet other embodiments, the stabilizingoligonucleotide and the mRNA polynucleotide transcript are contacted atabout a 0.5:1 ratio. In still other embodiments, the stabilizingoligonucleotide and mRNA polynucleotide transcript are contacted atabout a 1:1 ratio. In certain embodiments, the stabilizingoligonucleotide and the mRNA polynucleotide transcript are contacted atabout a 2:1, 5:1, a 10:1, a 100:1 or a 1,000:1 ratio.

Upon contacting the mRNA polynucleotide transcript with a complementarystabilizing oligonucleotide, the stabilizing oligonucleotide willhybridize to the mRNA polynucleotide (e.g., at a region ofcomplementarity). Upon hybridizing to the mRNA, the stabilizingoligonucleotide will form a duplexed region with, for example, thenon-coding region of the mRNA polynucleotide and thereby render the mRNApolynucleotide more resistant to nuclease degradation. As a result ofbeing rendered more resistant to nuclease (e.g., endonuclease)degradation, the amount of the expression product (e.g., a polypeptide)translated from the mRNA polynucleotide transcript may be increased(e.g., increased by at least about 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%,30%, 33%, 36%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 110%,120%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 750%,800%, 900%, 1,000% or more). In certain embodiments, one or both of themRNA transcript or the stabilizing oligonucleotide may comprise at leastone modification (e.g., one or more chemically modified nucleobases ormodified inter-nucleotide bonds).

Also provided herein are methods of increasing the translationalefficiency of an exogenous mRNA transcript. Such methods may facilitate,for example, an increase in the production of an expression productproduced following translation of the mRNA polynucleotides ortranscripts of the present inventions. Generally, such methods comprisea step of contacting the mRNA polynucleotide transcript with astabilizing oligonucleotide that is complementary to the coding and/ornon-coding region of the mRNA transcript under suitable conditions(e.g., high stringency conditions), thereby causing the mRNApolynucleotide transcript and the stabilizing oligonucleotide tohybridize to each other. Such methods may be employed to render the mRNAtranscript more resistant to nuclease (e.g., exonuclease) degradationwhile modulating (e.g., increasing) the translational efficiency of theexogenous mRNA transcript by one or more target cells. In certainembodiments, the stabilizing oligonucleotide comprises at least onemodified nucleobase. In certain embodiments, the mRNA transcript alsocomprises one or more modifications (e.g., one or more chemicalmodifications and/or phosphorothioate inter-nucleosidic bonds).

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of the present invention whereby anmRNA polynucleotide transcript (as indicated by

) having a poly-A tail located downstream (3′) of the coding region iscontacted with a 15-mer poly(2′O-Me-uracil) stabilizing oligonucleotidehaving a phosphorothioate backbone. As illustrated, upon contacting thepoly-A tail of the mRNA polynucleotide with the fully complementarystabilizing oligonucleotide a duplexed region is formed and therebystabilizes the mRNA polynucleotide by rendering it more resistant tonuclease degradation. Because the stabilizing oligonucleotide is fullycomplementary to multiple regions of the depicted poly-A tail, thereexist several possible duplexed constructions, only four of which areillustrated in the depicted embodiment.

FIG. 2 depicts the cumulative amount of erythropoietin protein (EPO)produced in vitro over a seventy-two hour period by 293T cellstransfected with various nuclease resistant polynucleotides of thepresent invention. Non-denatured human EPO mRNA was hybridized with a15-mer of fully phosphorothioated 2-OMe-uridine oligonucleotides in theratios listed (oligo:mRNA). The depicted plot is represented as apercentage of the protein produced from the unhybridized native EPOmRNA. As illustrated in FIG. 2 relative to the untreated EPO mRNApolynucleotide (designated “Unhybridized”), the stabilized EPOpolynucleotide mRNA transcripts generally demonstrated an increase inthe amount of EPO protein expressed by the 293T cells that weretransfected with the stabilized mRNA transcript, and in certaininstances an approximately 160% increase in the amount of EPO proteintranslated and produced was observed relative to the Unhybridizedcontrol.

FIG. 3 illustrates the quantification of cumulative human erythropoietin(EPO) protein produced in vitro over a seventy-two hour period by 293Tcells transfected with various stabilized mRNA transcripts of thepresent invention. Denatured human EPO mRNA was hybridized with a 15-merof fully phosphorothioated 2-OMe-uridine oligonucleotides in the ratioslisted (oligo:mRNA). The depicted plot is represented as a percentage ofthe protein produced from the unhybridized denatured EPO mRNA. Asillustrated in FIG. 3 , the stabilized EPO polynucleotide transcriptsgenerally yielded higher cumulative amounts of EPO protein translatedand produced by the 293T cells transfected with the stabilized mRNAtranscripts disclosed herein.

FIG. 4 depicts the cumulative amount of erythropoietin (EPO) proteinproduced in vitro by 293T cells transfected with various nucleaseresistant polynucleotides of the present invention at different timepoints over a ninety-six hour period. Human EPO mRNA was hybridized witha 30-mer of fully phosphorothioated 2-OMe-uridine oligonucleotides inthe ratios listed (oligo:mRNA). The depicted plot is represented as apercentage of the protein produced from the respective unhybridizednative EPO mRNA. As illustrated in FIG. 4 , relative to the untreatedEPO mRNA polynucleotide (designated “Unhybridized”), the stabilized ornuclease resistant polynucleotide mRNA transcripts generallydemonstrated an increase in the amount of EPO protein expressed by the293T cells transfected with such stabilized mRNA transcripts. Inparticular, those stabilized or nuclease resistant mRNA transcripts thatwere prepared by exposure of the mRNA transcript to lower concentrationsof stabilizing oligonucleotide (e.g., 0.1 and 0.5) demonstrated highertranslational efficiencies relative to their unmodified counterparts.

DETAILED DESCRIPTION

The present inventions are directed to stabilized or nuclease resistantpolynucleotides and compositions (e.g., mRNA polynucleotides) andrelated methods of their use and preparation. In certain embodiments thepolynucleotides and compositions disclosed herein encode one or morefunctional expression products (e.g., polypeptides, proteins and/orenzymes) and are not subject to some of the limitations that aregenerally associated with conventional gene or enzyme replacementtherapies. For example, in embodiments where the polynucleotidetranscripts disclosed herein comprise mRNA, such polynucleotides neednot integrate into a host cells' genome to exert their therapeuticeffect. Similarly, in certain embodiments, the exogenous polynucleotidetranscripts are translated by the host cells and accordingly arecharacterized by the native post-translational modifications that arepresent in the native expression product.

While the administration of exogenous polynucleotides (e.g., DNA or RNA)represents a meaningful advancement in the treatment diseases, theadministration of such exogenous polynucleotides is often hampered bythe limited stability of such polynucleotides, particularly followingtheir in vivo administration. For example, following theiradministration to a subject, many polynucleotides may be subject tonuclease (e.g., exonuclease and/or endonuclease) degradation. Nucleasedegradation may negatively influence the capability of an mRNApolynucleotide transcript to reach a target cell or to be translated,the result of which is to preclude the exogenous polynucleotide fromexerting an intended therapeutic effect.

Nucleases represent a class of enzymes that are responsible for thecleavage or hydrolysis of the phosphodiester bonds that hold thenucleotides of DNA or RNA together. Those nuclease enzymes that cleaveor hydrolyze the phosphodiester bonds of DNA are calleddeoxyribonucleases, while the nuclease enzymes that cleave thephosphodiester bonds of RNA are called ribonucleases. As generally usedherein, the term “nuclease” refers to an enzyme with the capability todegrade or otherwise digest polynucleotides or nucleic acid molecules(e.g., DNA or RNA). Representative examples of nucleases includeribonucleases (RNase) which digests RNA, and deoxyribonuclease (DNase)which digests DNA. Unless otherwise specified, the term “nuclease”generally encompasses nuclease enzymes that are capable of degradingsingle-stranded polynucleotides (e.g., mRNA) and/or double strandedpolynucleotides (e.g., DNA).

In certain aspects, the present invention is directed to methods andstrategies for stabilizing polynucleotides from nuclease degradation orfor improving the resistance of one or more polynucleotides (e.g., mRNA)to nuclease degradation. It should be noted that in certain embodiments,improvements in the stability and/or nuclease resistance of thepolynucleotides disclosed herein may be made with reference to a nativeor unmodified polynucleotide. For example, in certain embodiments, thestability and/or nuclease resistance of a polynucleotide (e.g., an mRNAtranscript) is increased by at least about 105%, 110%, 115%, 120%, 125%,130%, 135%, 140%, 150%, 160%, 170%, 175%, 180%, 190%, 200%, 250%, 300%,350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, or more relativeto the native or unmodified polynucleotide transcript.

As used herein to characterize a polynucleotide (e.g., an mRNAtranscript encoding a functional urea cycle enzyme), the term “stable”generally refers to a reduced susceptibility to degradation ordestruction (e.g., a reduced susceptibility to nuclease cleavage invivo). For example, the term “stable” may be used to refer to areduction in the rate of nuclease degradation of a polynucleotide invivo. In certain embodiments, the half-life (t½) of a polynucleotiderepresents an objective measurement of its stability. Similarly, incertain embodiments, the amount or mass of an expression product that isproduced following the expression (e.g., translation) of a stable ornuclease resistant polynucleotide represents an objective measurement ofits stability. Preferred are modifications made or otherwise introducedinto a polynucleotide that serve to enhance (e.g., increase) thehalf-life or translational efficiency of such polynucleotide in vivorelative to its unmodified counterpart. For example, in certainembodiments, the t½ of a nuclease resistant polynucleotide (e.g., anmRNA transcript) is increased by at least about 105%, 110%, 115%, 120%,125%, 130%, 135%, 140%, 150%, 160%, 170%, 175%, 180%, 190%, 200%, 250%,300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, or morerelative to its native or unmodified polynucleotide counterpart. Incertain embodiments, the stability of hybridized mRNA may be in part dueto the inherent single strand specificity of some nuclease enzymes, andin particular RNase enzymes.

The methods disclosed herein generally comprise a step of contacting thenon-coding region of the polynucleotide (e.g., the poly-A tail of anmRNA polynucleotide) with a complementary (e.g., a perfectlycomplementary) stabilizing oligonucleotide under suitable conditions,thereby causing the stabilizing oligonucleotide to hybridize to thenon-coding region of the polynucleotide. As used herein, the terms“contact” and “contacting” generally refer to bringing two or moremoieties together or within close proximity of one another such that themoieties may react. For example, in certain embodiments of the presentinvention, a polynucleotide (e.g., an mRNA transcript) may be contactedwith one or more stabilizing oligonucleotides (e.g., a stabilizingoligonucleotide that is perfectly complementary to a region or fragmentof the polynucleotide) such that the polynucleotide and stabilizingoligonucleotide would be expected to react (e.g., hybridize to oneanother) under suitable conditions.

Upon hybridizing of the stabilizing oligonucleotide (e.g., a 15-merpoly(2′-O-Me-uracil) oligonucleotide) to the polynucleotide, thepolynucleotide is rendered more resistant to nuclease degradation. Forexample, in certain embodiments, provided herein are methods ofincreasing the nuclease resistance of an mRNA polynucleotide comprisinga poly-A tail by contacting the poly-A tail of such polynucleotide witha complementary poly-U stabilizing oligonucleotide. Upon hybridizing tothe non-coding region of the polynucleotide (e.g., the poly-A tail) toform a duplexed or double-stranded region, nuclease degradation of thepolynucleotide may be reduced, delayed or otherwise prevented. Withoutwishing to be bound by any particular theories, it is believed that theobserved stability and nuclease resistance of the polynucleotidesdisclosed herein is due in part to the single-stranded specificity ofsome nuclease enzymes (e.g., ribonucleases). In those embodiments whereone or both of the stabilizing oligonucleotide and/or the polynucleotidecomprise a modification (e.g., a chemically-modified nucleobases and/ora phosphorothioate backbone) such modifications may serve to furtherstabilize the polynucleotide by sterically interfering with nucleasedegradation.

It should be noted that while the terms “polynucleotide” and“oligonucleotide” may be generally understood by those of ordinary skillin the art to generally be synonymous with each other, such terms areused herein for convenience to distinguish the targeted sense nucleicacid transcripts (e.g., mRNA) from the shorter (e.g., about 15-50 mer)complementary or anti-sense nucleic acids that are used to modulate thestability of a targeted sense nucleic acid transcript in accordance withthe teachings of the present inventions. In particular, the phrase“stabilizing oligonucleotide” is used herein to describe a nucleic acidsequence that is generally complementary or anti-sense to a region orfragment of a polynucleotide sequence encoding a functional expressionproduct. While such stabilizing oligonucleotides may generally be of anylength, in certain embodiments the stabilizing oligonucleotides are lessthan 500 nucleotides, less than 400 nucleotides, less than 300nucleotides, less than 250 nucleotides, less than 200 nucleotides, lessthan 100 nucleotides, or more preferably less than 50 nucleotides, lessthan 40 nucleotides, less than 30 nucleotides, less than 25 nucleotides,less than 20 nucleotides, less than 19 nucleotides, less than 18nucleotides, less than 17 nucleotides, less than 16 nucleotides or lessthan 15 nucleotides in length.

In certain embodiments, the stabilizing oligonucleotides (e.g., a 15-merpoly-U stabilizing oligonucleotide) disclosed herein comprise one ormore modifications (e.g., modifications such as 2′-O-alkyl sugarsubstitutions). For example, in some embodiments the stabilizingoligonucleotide comprises one or more chemical modifications, such asone or more 2′-O-alkyl modified or substituted nucleobases or theinclusion of one or more phosphorothioate inter-nucleobase linkages.Such modifications may further improve the ability of the stabilizingoligonucleotide to hybridize to a complementary polynucleotide or mayimprove the stability or nuclease resistance of such polynucleotide(e.g., by interfering with recognition of such polynucleotide bynuclease enzymes).

The present inventors have surprisingly discovered that stabilized mRNApolynucleotides that were prepared by exposure of the mRNApolynucleotides to higher concentrations of stabilizing oligonucleotidesresulted in the production of lower quantities of the encoded expressionproduct (e.g., erythropoietin protein) by cells transfected with suchpolynucleotides. Without wishing to be bound by a particular theory, itis suspected that higher degrees of hybridization of the stabilizingoligonucleotides to the polynucleotide may interfere with the ability ofthe resulting duplexed (i.e., hybridized or stabilized) polynucleotideto form secondary or even tertiary structures (e.g., hairpin loops,bulges, and internal loops) that may also contribute to the stability ofsuch polynucleotide. For example, while higher degrees of hybridizationof the poly-A tail region of an mRNA polynucleotide transcript mayimprove the nuclease resistance of such mRNA transcript, the longerduplexed regions may also interfere with the ability of the duplexedmRNA transcript to properly fold. In certain instances where the properfolding of such mRNA transcript contributes to its stability (e.g.,nuclease resistance), it is expected that interference with the abilityof such transcript to properly fold may be associated with acorresponding reduction in its stability. Accordingly, in certainembodiments, shorter stabilizing oligonucleotides (e.g., about 75, 70,60, 65, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5 nucleotides or less) are preferred.Similarly, in certain embodiments the hybridization of a stabilizingoligonucleotide to a polynucleotide does not materially interfere withthe ability of the resulting nuclease resistant polynucleotide to formsecondary or tertiary structures.

The degree to which the nuclease resistant polynucleotides disclosedherein hybridize may be a direct function of the manner in which suchnuclease resistant polynucleotides were prepared. As depicted in FIG. 1, to the extent that an mRNA polynucleotide is contacted with a highconcentration of a complementary stabilizing oligonucleotide, thestabilizing oligonucleotide may hybridize to the mRNA polynucleotide atmultiple regions. In certain embodiments, the extent to which apolynucleotide hybridizes with a complementary stabilizingoligonucleotide may be manipulated or otherwise controlled by modifyingthe relative concentrations of stabilizing oligonucleotide to which thepolynucleotide is exposed. For example, in certain preferredembodiments, the stabilizing oligonucleotide and the mRNA polynucleotidetranscript are contacted at about a 0.1:1 ratio. In other embodiments,the stabilizing oligonucleotide and the mRNA polynucleotide transcriptare contacted at about a 0.25:1 ratio. In yet other embodiments, thestabilizing oligonucleotide and the mRNA polynucleotide transcript arecontacted at about a 0.5:1 ratio. In still other embodiments, thestabilizing oligonucleotide and mRNA polynucleotide transcript arecontacted at about a 1:1 ratio. In certain embodiments, the stabilizingoligonucleotide and the mRNA polynucleotide transcript are contacted atabout a 5:1, a 10:1, a 100:1 or a 1,000:1 ratio.

As used herein, the term “polynucleotide” is generally used to refer toa nucleic acid (e.g., DNA or RNA) to be stabilized or rendered morenuclease resistant in accordance with the teachings of the presentinvention. In certain embodiments, the polynucleotides disclosed herein(or particular regions or fragments thereof) represent the nucleic acidtarget to which the stabilizing oligonucleotides may hybridize. Thepolynucleotides (e.g., an mRNA polynucleotide) disclosed herein may alsocomprise one or more modifications. For example, in some embodiments themRNA polynucleotide transcripts disclosed herein comprise one or morechemical modifications, which in certain instances may further improvethe stability or nuclease resistance of such polynucleotide transcript(e.g., by sterically hindering or otherwise interfering with nucleasedegradation).

The polynucleotides may comprise both coding and non-coding regions andin certain embodiments described herein, the stabilizingoligonucleotides hybridize to the non-coding region of thepolynucleotide. As used herein, the phrase “non-coding region” generallyrefers to that portion or region of the polynucleotide or a gene that isnot a coding region and that is not expressed, transcribed, translatedor otherwise processed into an expression product such as an amino acid,polypeptide, protein or enzyme. In the context of DNA polynucleotides,the non-coding region may comprise intron sequences or other sequenceslocated 5′ or 3′ (e.g., upstream or downstream) of the coding region(e.g., promoters, enhancers, silencers). In the context of RNApolynucleotides, the non-coding region may comprise sequences located 5′or 3′ (e.g., upstream or downstream) of the coding region (e.g., 3′untranslated region (UTR), a 5′ untranslated region (UTR), a poly-A tailand a terminal cap). In certain embodiments, the targeted non-codingregion may comprise two distinct, but overlapping regions. For example,as briefly depicted below a stabilizing oligonucleotide may be preparedsuch that it is perfectly complementary to a region of a polynucleotidecomprising or spanning a fragment of the 3′ untranslated region (UTR)and a fragment of the poly-A tail.

mRNA Polynucleotide Fragment: 5′-....AUGGCACAUCCUGUAAAAAAAAAAAAAAAAAAAAA...-3′                                                  |||||||||||||||||Stabilizing Oligonucleotide:  3′-                 CAUUUUUUUUUUUUUUU       -5′

Similarly, a stabilizing oligonucleotide may be prepared such that it iscomplementary to a region of a polynucleotide comprising or spanning afragment of a 5′ cap structure and a fragment of the 5′ UTR. Forexample, the stabilizing oligonucleotide may be complementary (e.g., atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%,95%, 97%, 98%, 99% or 100% complementary) to one or more non-codingregions of the polynucleotide selected from the group of regionsconsisting of the 5′ UTR, a 5′ terminal cap, the 3′ UTR and the poly-Atail. In certain embodiments, the hybridization of a complementarystabilizing oligonucleotide to the non-coding region of an mRNApolynucleotide is preferred, in part due to concerns relating to theability of the resultant duplexed region (i.e., the hybridizedpolynucleotide and stabilizing oligonucleotide) to interfere with thetranslation of the coding region.

As used herein, the phrase “coding region” generally refers to thatportion or region of the polynucleotide or a gene that when expressed,transcribed, translated or otherwise processed results in the productionof an expression product, such as an amino acid, polypeptide, protein orenzyme. It should be understood that while certain embodiments disclosedherein contemplate the hybridization of complementary stabilizingoligonucleotides to the non-coding region of a polynucleotidetranscript, the present invention need not be limited to suchembodiments. Rather, the present invention also contemplates thehybridization of the complementary stabilizing oligonucleotides toregions of the polynucleotide transcript (e.g., mRNA) comprising orspanning both the coding and non-coding regions. For example, astabilizing oligonucleotide may be prepared such that it targets and/oris complementary (e.g., perfectly complementary) to a fragment of thecoding region of an mRNA polynucleotide transcript and a fragment of thenon-coding 3 UTR located downstream of the coding region. The foregoingtherefore provides a means of specifically targeting a particular regionof the polynucleotide, such as the region located immediately downstreamof the coding region. Additionally, the foregoing also provides means ofcontrolling or otherwise affecting the degree to which a stabilizingoligonucleotide hybridizes to a complementary region of thepolynucleotide. In certain embodiments where the stabilizingoligonucleotide targets the coding region (or a fragment thereof)preferably the hybridization of the stabilizing oligonucleotide to suchcoding region (or fragment thereof) does not interfere with theexpression (e.g., transcription or translation) of such polynucleotide.Similarly, in embodiments where the stabilizing oligonucleotide targetsthe coding region (or a fragment thereof) preferably the hybridizationof the stabilizing oligonucleotide to such coding region (or fragmentthereof) does not substantially interfere with the expression (e.g.,transcription or translation) of such polynucleotide.

In the context of the present invention the term “expression” is used inits broadest sense to refer to either the transcription of a specificpolynucleotide (e.g., a gene or nucleic acid) into an RNA transcript, orthe translation of at least one mRNA polynucleotide into a polypeptide,protein or enzyme. For example, disclosed herein are compositions whichcomprise one or more mRNA polynucleotides that encode functionalexpression products (e.g., proteins or enzymes), and in the context ofsuch mRNA polynucleotides, the term expression refers to the translationof such mRNA polynucleotides to produce a polypeptide, protein or enzymeencoded thereby. Similarly, the phrase “expression product” is usedherein in its broadest sense to generally refer to an RNA transcriptionproduct that is transcribed from a DNA polynucleotide, or alternativelyto a polypeptide, protein or enzyme that is the natural translationproduct of an mRNA polynucleotide. In certain embodiments, theexpression product of the polynucleotide is a functional enzyme (e.g., aurea cycle enzyme). In certain embodiments, the expression product ofthe polynucleotide is a functional protein (e.g., hormone) or enzyme. Inthose instances where the polynucleotide is DNA, following expression(i.e., transcription) of such DNA the encoded expression product (i.e.,RNA) may be produced. Similarly, in those embodiments where thepolynucleotide is mRNA, following expression (i.e., translation) of suchmRNA the encoded expression product (e.g., a polypeptide, protein orenzyme) may be produced and/or excreted.

In some embodiments, the present inventions are directed to methods ofmodulating (e.g., increasing, improving or otherwise enhancing) thetranslational efficiency of one or more mRNA polynucleotides in a targetcell. As used herein, the phrase “translational efficiency” refers tothe rate at which an mRNA polynucleotide is translated and thecorresponding expression product produced. In certain instances, thestable or nuclease resistant mRNA polynucleotides disclosed herein maybe characterized by their increased translational efficiency, resultingin a corresponding increase in the production of the expression productencoded by such mRNA polynucleotide. Such methods generally comprise aninitial step of contacting an mRNA polynucleotide with a complementary(e.g., perfectly or partially complementary) stabilizing oligonucleotideunder suitable conditions (e.g., high stringency conditions), therebycausing the mRNA polynucleotide and one or more stabilizingoligonucleotides to hybridize to each other. As a result, the mRNApolynucleotide is rendered more resistant to nuclease degradation andthe translational efficiency of such polynucleotide in one or moretarget cells increased. In certain embodiments, one or both of thestabilizing oligonucleotide and/or the mRNA polynucleotide may compriseat least one modified nucleobase (e.g., a 2′-O-alkyl sugarsubstitution). In certain embodiments, one or both of the stabilizingoligonucleotide and the mRNA polynucleotide also comprise one or moremodifications (e.g., one or more nucleobases linked by phosphorothioatebonds).

In certain instances, the nuclease resistant polynucleotides disclosedherein may be recombinantly-prepared (e.g., a recombinantly-preparedcodon-optimized mRNA polynucleotide). In such embodiments, suchpolynucleotides (e.g., a recombinantly-prepared mRNA polynucleotide) maybe contacted with a complementary stabilizing oligonucleotide prior tobeing administered to a subject in a suitable carrier or vehicle (e.g.,a lipid nanoparticle).

Also contemplated by the present invention is the direct administrationof an exogenous stabilizing oligonucleotide to a subject (e.g., for thetreatment of a disease or condition associated with the suboptimal orsub-therapeutic production of an expression product, such as a proteinor enzyme). In such embodiments, the present inventions provide a meansof modulating (e.g., increasing or otherwise enhancing) the expression,production and/or secretion of an endogenous expression product. Forexample, the present inventions contemplate the administration of astabilizing oligonucleotide to a subject, wherein the stabilizingoligonucleotide is complementary (e.g., perfectly- orpartially-complementary) to an endogenous polynucleotide (e.g., mRNA).In such embodiments, an exogenously-prepared stabilizing oligonucleotidethat is complementary (e.g., perfectly complementary) to a region of anendogenous polynucleotide (e.g., the non-coding region of an endogenousmRNA polynucleotide) is administered to a subject. Following theadministration of the exogenous stabilizing oligonucleotide, sucholigonucleotide hybridizes to the one or more endogenous polynucleotides(e.g., mRNA) encoding an under-expressed expression product such thatthe stability or the nuclease resistance of the endogenouspolynucleotide is modulated (e.g., enhanced or otherwise increased)and/or its translational efficiency increased. The resulting stabilizedor nuclease resistant endogenous polynucleotide (e.g., mRNA) may becharacterized as having an increase circulatory half-life (t_(1/2))relative to its native counterpart and, in certain instances an improvedtranslational efficiency. As a result, the amount of the expressionproduct (e.g., a lysosomal enzyme) encoded by such endogenouspolynucleotides may be enhanced or otherwise increased and an underlyingcondition (e.g., a protein or enzyme deficiency) or its symptoms therebytreated or mitigated. The foregoing therefore provides a means ofincreasing the expression of sub-optimally expressed endogenous mRNApolynucleotides by rendering such polynucleotides more nucleaseresistant relative to their native (and under-expressed) counterparts.It should be understood that while the foregoing embodiments (i.e., thedirect administration of stabilizing oligonucleotides to a subject) maygenerally relate to traditional anti-sense or RNAi mechanisms oftargeting endogenous nucleic acids mRNA), the observed effect of suchtargeting is an increase, rather than a decrease, in the production ofthe expression product encoded by the targeted polynucleotide. Incertain embodiments, the stabilizing oligonucleotide is delivered oradministered to a subject in a suitable pharmaceutical carrier, vehicleor composition (e.g., encapsulated in a lipid nanoparticle vehicle).

The polynucleotides provided herein, and in particular the mRNApolynucleotides provided herein, preferably retain at least some abilityto be expressed or translated, to thereby produce a functionalexpression product (e.g., a protein or enzyme) within a target cell.Accordingly, the present invention also relates to the administration ofstabilized or duplexed polynucleotides to a subject (e.g., mRNA whichhas been stabilized against in vivo nuclease digestion or degradation).In a preferred embodiment of the present invention, the therapeuticactivity of the nuclease resistant polynucleotide is prolonged orotherwise evident over an extended period of time (e.g., at least abouttwelve hours, twenty-four hours, thirty-six hours, seventy-two hours,four days, five days, 1 week, ten days, two weeks, three weeks, fourweeks, six weeks, eight weeks, ten weeks, twelve weeks or longer). Forexample, the therapeutic activity of the nuclease resistantpolynucleotides may be prolonged such that the compositions of thepresent invention are administered to a subject on a semi-weekly orbi-weekly basis, or more preferably on a monthly, bi-monthly, quarterlyor even on an annual basis. The extended or prolonged activity of thecompositions of the present invention, and in particular of the nucleaseresistant mRNA polynucleotides comprised therein, is directly related tothe translational efficiency of such polynucleotide and the quantity ofthe expression product (e.g., a functional protein or enzyme) that canbe translated from such mRNA.

In certain embodiments the translational efficiency and the in vivoactivity of the nuclease resistant polynucleotides and compositions ofthe present invention may be further extended or prolonged by theintroduction of one or more modifications to such polynucleotides toimprove or enhance their half-life (t½). For example, the Kozacconsensus sequence plays a role in the initiation of proteintranslation, and the inclusion of such a Kozac consensus sequence in themRNA polynucleotides of the present invention may further extend orprolong the activity or translational efficiency of such mRNApolynucleotides. Furthermore, the quantity of functional protein orenzyme translated by the target cell is a function of the quantity ofpolynucleotide (e.g., mRNA) delivered to the target cells and thestability of such polynucleotide. To the extent that the stabilityand/or half-life of the nuclease resistant polynucleotides of thepresent invention may be improved or enhanced, the therapeutic activityof the translated protein or enzyme and/or the dosing frequency of thecomposition may be further extended.

Accordingly, in a preferred embodiment, one or both of thepolynucleotides and/or the stabilizing oligonucleotides disclosed hereincomprise at least one modification. As used herein, the terms“modification” and “modified” as they relate to the polynucleotidesand/or stabilizing oligonucleotides provided herein, refer to at leastone alteration or chemical modification introduced into suchpolynucleotides and/or stabilizing oligonucleotides and which preferablyrenders them more stable (e.g., resistant to nuclease digestion) thanthe wild-type or naturally occurring version of the polynucleotide. Forexample, the introduction of chemical modifications into one or more ofthe polynucleotide and the stabilizing oligonucleotide may interferewith, sterically hinder or otherwise delay their recognition and/ordegradation by one or more nuclease enzymes (e.g., RNase). Increasedstability can include, for example, less sensitivity to hydrolysis orother destruction by endogenous enzymes (e.g., endonucleases orexonucleases) or conditions within the target cell or tissue, therebyincreasing or enhancing the circulatory half-life or residence time ofsuch polynucleotides in the target cell, tissue, subject and/orcytoplasm. The stabilized or nuclease resistant polynucleotides providedherein may demonstrate longer half-lives relative to their naturallyoccurring or un-hybridized counterparts (e.g. the wild-type version ofthe polynucleotide). Also contemplated by the terms “modification” and“modified”, as such terms relate to mRNA polynucleotides and/orstabilizing oligonucleotides of the present invention, are alterationswhich improve or enhance the translational efficiency of such mRNApolynucleotides, including for example, the inclusion of sequences whichaffect the initiation of protein translation (e.g., the Kozac consensussequence). (See, Kozak, M., Nucleic Acids Res. (1987); 15 (20):8125-48).

Exemplary modifications to a polynucleotide may also include thedepletion of a base (e.g., by deletion or by the substitution of onenucleotide for another) or modification of a base, for example, thechemical modification of a base. The phrase “chemical modifications” asused herein, includes modifications which introduce chemistries thatdiffer from those observed in naturally occurring polynucleotides, forexample, covalent modifications such as the introduction of modifiedbases (e.g., nucleotide analogs, or the inclusion of pendant groupswhich are not naturally found in such polynucleotides). In certainembodiments, exemplary chemical modifications that may be introducedinto one or both of the polynucleotide and the stabilizingoligonucleotide include pseudouridine, 2-thiouracil, 5-methyl cytidine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil.5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurineand 2-chloro-6-aminopurine cytosine.

In addition, suitable modifications may include alterations in one ormore nucleotides of a codon such that the codon encodes the same aminoacid but is more stable relative to the wild-type codon of thepolynucleotide found in nature. For example, an inverse relationshipbetween the stability of RNA and a higher number cytidines (C) and/oruridines (U) residues has been demonstrated, and RNA lacking C and Uresidues have been found to be stable to most RNases. (Heidenreich, etal. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number ofC and/or U residues in an mRNA sequence is reduced. In otherembodiments, the number of C and/or U residues is reduced bysubstitution of one codon encoding a particular amino acid for anothercodon encoding the same or a related amino acid. Contemplatedmodifications to the mRNA polynucleotides of the present invention alsoinclude the incorporation of pseudouridines. The incorporation ofpseudouridines into the mRNA polynucleotides of the present inventionmay enhance their stability and translational capacity, as well asdiminish their immunogenicity in vivo. (See, e.g., Kariko, K., et al.,Molecular Therapy 16 (11): 1833-1840 (2008)). Substitutions andmodifications to the polynucleotides of the present invention may beperformed by methods readily known to one or ordinary skill in the art.

The constraints on reducing the number of C and U residues in a sequencewill likely be greater within the coding region of an mRNApolynucleotide, compared to its untranslated region, (i.e., it willlikely not be possible to eliminate all of the C and U residues presentin the coding region while still retaining the ability of the message toencode the desired amino acid sequence). The degeneracy of the geneticcode, however presents an opportunity to allow the number of C and/or Uresidues that are present in the sequence to be reduced, whilemaintaining the same coding capacity (i.e., depending on which aminoacid is encoded by a codon, several different possibilities formodification of RNA sequences may be possible). For example, the codonsfor Gly can be altered to GGA or GGG instead of GGU or GGC.

As previously mentioned, the term modification also includes, forexample, the incorporation of non-nucleotide linkages or modifiednucleotides into the polynucleotides and/or stabilizing oligonucleotidesof the present invention. Such modifications include the addition ofbases to a polynucleotide sequence (e.g., the inclusion of a poly-A tailor the lengthening of the poly-A tail), the alteration of the 3′ UTR orthe 5′ UTR, and the inclusion of elements which change the structure ofa polynucleotide and/or stabilizing oligonucleotide (e.g., elementswhich modulate the ability of such polynucleotides or their expressionproducts to form secondary structures).

In certain embodiments the poly-A tail and the region immediatelyupstream represent suitable regions of a polynucleotide that thestabilizing oligonucleotides (e.g., a 15-mer poly-U stabilizingoligonucleotide) disclosed herein may target and/or hybridize to. Thepoly-A tail is thought to naturally stabilize natural mRNApolynucleotides and synthetic sense RNA. Therefore, in certainembodiments a long poly-A tail can be added to an mRNA polynucleotideand thus render the mRNA more stable. In other embodiments, the poly-Atail or a particular region thereof may be contacted under suitablecondition (e.g., high stringency conditions) with a complementarystabilizing oligonucleotide (e.g., a poly-U stabilizing oligonucleotide)and thereby render the polynucleotide more nuclease resistant. Poly-Atails can be added using a variety of art-recognized techniques. Forexample, long poly-A tails can be added to synthetic or in vitrotranscribed RNA using poly-A polymerase. (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). In addition, poly-A tails can beadded by transcription directly from PCR products or may be ligated tothe 3′ end of an mRNA polynucleotide with RNA ligase. (See, e.g.,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)). Incertain embodiments, the length of the poly-A tail is at least about 20,40, 50, 75, 90, 100, 150, 200, 250, 300, 350, 400, 450 or at least 500nucleotides. In certain embodiments, the length of the poly-A tail isadjusted to control the stability of an mRNA polynucleotide of theinvention. For example, since the length of the poly-A tail caninfluence the half-life of an mRNA polynucleotide, the length of thepoly-A tail can be adjusted to modify the level of resistance of themRNA to nucleases and thereby control its translational efficiency in atarget cell. In certain embodiments, the stabilized or nucleaseresistant polynucleotides are sufficiently resistant to in vivodegradation (e.g., by nucleases), such that they may be delivered to thetarget cell without a carrier.

In certain embodiments, a polynucleotide can be modified by theincorporation 3′ and/or 5′ untranslated (UTR) sequences which are notnaturally found in the wild-type polynucleotide. In certain embodiments,3′ and/or 5′ flanking sequences which naturally flank an mRNA and encodea second, unrelated protein can be incorporated into the nucleotidesequence of an mRNA polynucleotide in order to further enhance itstranslational efficiency. For example, 3′ or 5′ sequences from mRNApolynucleotides which are stable (e.g., globin, actin, GAPDH, tubulin,histone, or citric acid cycle enzymes) can be incorporated into the 3′and/or 5′ region of a sense mRNA polynucleotide to increase itsstability. To the extent such modifications are incorporated into apolynucleotide, in certain embodiments the regions of the polynucleotideincluding such modifications (e.g., a 3′ UTR) may also represent asuitable target to which the stabilizing oligonucleotides disclosedherein may hybridize to in an effort to further stabilize such modifiedpolynucleotide.

The present inventions also contemplate modifications to the 5′ end ofthe polynucleotides (e.g., mRNA) to include a partial sequence of a CMVimmediate-early 1 (IE1) gene, or a fragment thereof (e.g., SEQ ID NO: 1or SEQ ID NO: 2) to improve the nuclease resistance and/or improve thehalf-life of the polynucleotide. In addition to increasing the stabilityof the mRNA polynucleotide sequence, it has been surprisingly discoveredthat the inclusion of a partial sequence of a CMV immediate-early 1(IE1) gene enhances the translation of the mRNA and the expression ofthe functional protein or enzyme. Also contemplated is the inclusion ofa sequence encoding human growth hormone (hGH), or a fragment thereof(e.g., SEQ ID NO: 3) to one or both of the 3′ and 5′ ends of thepolynucleotide (e.g., mRNA) to further stabilize the polynucleotide.Generally, preferred modifications improve the stability, translationalefficiency, nuclease resistance and/or pharmacokinetic properties (e.g.,half-life) of the polynucleotide relative to its unmodified counterpart,and include, for example modifications made to improve suchpolynucleotide's resistance to in vivo nuclease digestion.

The administration of the compositions, stabilized polynucleotides andstabilizing oligonucleotides disclosed herein may be facilitated byformulating such compositions in a suitable carrier (e.g., a lipidnanoparticle). As used herein, the term “carrier” includes any of thestandard pharmaceutical carriers, vehicles, diluents, excipients and thelike which are generally intended for use in connection with theadministration of biologically active agents, including polynucleotides.The compositions and in particular the carriers described herein arecapable of delivering polynucleotides and/or stabilizingoligonucleotides of varying sizes to their target cells or tissues. Incertain embodiments of the present invention, the carriers of thepresent invention are capable of delivering large polynucleotidesequences (e.g., polynucleotides of at least 1 kb, 1.5 kb, 2 kb, 2.5 kb,5 kb, 10 kb, 12 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50kb, or more). The polynucleotides can be formulated with one or moreacceptable reagents to facilitate the delivery of such polynucleotidesto target cells. Appropriate reagents are generally selected withregards to a number of factors, which include, among other things, thebiological or chemical properties of the polynucleotides (e.g., charge),the intended route of administration, the anticipated biologicalenvironment to which such polynucleotides will be exposed and thespecific properties of the intended target cells. In some embodiments,carriers, such as liposomes or synthetically-prepared exosomes,encapsulate the polynucleotides. In some embodiments, the carrierdemonstrates preferential and/or substantial binding to a target cellrelative to non-target cells. In a preferred embodiment, the carrierdelivers its contents to the target cell such that the polynucleotidesare delivered to the appropriate subcellular compartment, such as thecytoplasm.

In certain embodiments, the carriers disclosed herein comprise aliposomal vesicle, or other means to facilitate the transfer of apolynucleotide to target cells and tissues. Suitable carriers include,but are not limited to, liposomes, nanoliposomes, ceramide-containingnanoliposomes, proteoliposomes, both natural and synthetically-derivedexosomes, natural, synthetic and semi-synthetic lamellar bodies,nanoparticulates, calcium phosphor-silicate nanoparticulates, calciumphosphate nanoparticulates, silicon dioxide nanoparticulates,nanocrystalline particulates, semiconductor nanoparticulates,poly(D-arginine), nanodendrimers, starch-based delivery systems,micelles, emulsions, niosomes, plasmids, viruses, calcium phosphatenucleotides, aptamers, peptides and other vectorial tags. Alsocontemplated is the use of bionanocapsules and other viral capsidproteins assemblies as a suitable carrier. (Hum. Gene Ther. 2008September; 19(9):887-95).

In a preferred embodiment of the present invention, the carrier isformulated as a lipid nanoparticle. As used herein, the phrase “lipidnanoparticle” refers to a carrier comprising one or more lipids (e.g.,cationic and/or non-cationic lipids). Preferably, the lipidnanoparticles are formulated to deliver one or more polynucleotides(e.g., mRNA) to one or more target cells or tissues. The use of lipids,either alone or as a component of the carrier, and in particular lipidnanoparticles, is preferred. Examples of suitable lipids include, forexample, the phosphatidyl compounds (e.g., phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides). Also contemplated is theuse of polymers as carriers, whether alone or in combination with othercarriers. Suitable polymers may include, for example, polyacrylates,polyalkycyanoacrylates, polylactide, polylactide-polyglycolidecopolymers, polycaprolactones, dextran, albumin, gelatin, alginate,collagen, chitosan, cyclodextrins and polyethylenimine. In certainembodiments, the carrier is selected based upon its ability tofacilitate the transfection of a target cell with one or morepolynucleotides.

In certain embodiments of the present invention, the carrier may beselected and/or prepared to optimize delivery of the polynucleotide tothe target cell, tissue or organ. For example, if the target cell is apneumocyte the properties of the carrier (e.g., size, charge and/or pH)may be optimized to effectively deliver such carrier to the target cellor organ, reduce immune clearance and/or promote retention in thattarget organ. Alternatively, if the target tissue is the central nervoussystem (e.g., to facilitate delivery of mRNA polynucleotides to targetedbrain or spinal tissue) selection and preparation of the carrier mustconsider penetration of, and retention within the blood brain barrierand/or the use of alternate means of directly delivering such carrier tosuch target tissue. In certain embodiments, the compositions of thepresent invention may be combined with agents that facilitate thetransfer of exogenous polynucleotides from the local tissues or organsinto which such compositions were administered to one or more peripheraltarget organs or tissues.

The use of liposomal carriers to facilitate the delivery ofpolynucleotides to target cells is contemplated by the presentinvention. Liposomes (e.g., liposomal lipid nanoparticles) are generallyuseful in a variety of applications in research, industry, and medicine,particularly for their use as carriers of diagnostic or therapeuticcompounds in vivo (Lasic, Trends Biotechnol., 16: 307-321, 1998;Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are usuallycharacterized as microscopic vesicles having an interior aqua spacesequestered from an outer medium by a membrane of one or more bilayers.Bilayer membranes of liposomes are typically formed by amphiphilicmolecules, such as lipids of synthetic or natural origin that comprisespatially separated hydrophilic and hydrophobic domains (Lasic, TrendsBiotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes canalso be formed by amphiphilic polymers and surfactants (e.g.,polymerosomes, niosomes, etc.).

In the context of the present invention, a liposomal carrier typicallyserves to transport the polynucleotide and/or stabilizingoligonucleotide to a target cell. For the purposes of the presentinvention, the liposomal carriers are prepared to contain the desiredpolynucleotides. The process of incorporating a desired compound (e.g.,a stabilized or nuclease resistant polynucleotide and/or a stabilizingoligonucleotide) into a liposome is often referred to as “loading”(Lasic, et al., FEBS Lett., 312: 255-258, 1992). Theliposome-incorporated polynucleotides may be completely or partiallylocated in the interior space of the liposome, within the bilayermembrane of the liposome, or associated with the exterior surface of theliposome membrane. The incorporation of a polynucleotide into liposomesis also referred to herein as “encapsulation” wherein the polynucleotideis entirely contained within the interior space of the liposome.

One primary purpose of incorporating a polynucleotide into a carrier,such as a liposome, is to protect the polynucleotide from an environmentwhich may contain enzymes (e.g., nuclease enzymes) or chemicals thatdegrade or otherwise negatively influence the stability of thepolynucleotides encapsulated therein. Accordingly, in a preferredembodiment of the present invention, the selected carrier is capable offurther enhancing the stability of the nuclease resistantpolynucleotides (e.g., a nuclease resistant mRNA polynucleotide encodinga functional protein) contained therein. For example, a liposomalcarrier may allow the encapsulated polynucleotide to reach the targetcell and/or may preferentially allow the encapsulated polynucleotide toreach the target cell, or alternatively limit the delivery of suchpolynucleotides to other sites or cells where the presence of theadministered polynucleotide may be useless or undesirable. Furthermore,incorporating the polynucleotides into a carrier, such as for example, acationic liposome, also facilitates the delivery of such polynucleotidesinto a target cell.

Ideally, liposomal carriers are prepared to encapsulate one or moredesired polynucleotides (e.g., a nuclease resistant mRNA polynucleotideencoding a urea cycle enzyme) such that the compositions demonstrate ahigh transfection efficiency, enhanced stability and improvedtranslational efficiency. While liposomes can facilitate theintroduction of polynucleotides into target cells, the addition ofpolycations (e.g., poly L-lysine and protamine), as a copolymer canfurther facilitate, and in some instances markedly enhance thetransfection efficiency of several types of cationic liposomes by 2-28fold in a number of cell lines both in vitro and in vivo. (See N. J.Caplen, et al., Gene Ther. 1995; 2: 603; S. Li, et al., Gene Ther. 1997;4, 891.)

The present invention contemplates the use of cationic lipids andliposomes to encapsulate and/or enhance the delivery of the nucleaseresistant polynucleotides and/or stabilizing oligonucleotides disclosedherein into their target cells and tissues. As used herein, the phrase“cationic lipid” refers to any of a number of lipid species that carry anet positive charge at a selected pH, such as physiological pH. Thecontemplated liposomal carriers and lipid nanoparticles may be preparedby including multi-component lipid mixtures of varying ratios employingone or more cationic lipids, non-cationic lipids and PEG-modifiedlipids. Several cationic lipids have been described in the literature,many of which are commercially available. In some embodiments, thecationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride or “DOTMA” is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84,7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated alone orcan be combined with a neutral lipid, such as, e.g.,dioleoylphosphatidylethanolamine or “DOPE” or other cationic ornon-cationic lipids into a liposomal carrier or a lipid nanoparticle,and such liposomes can be used to enhance the delivery ofpolynucleotides into target cells. Particularly suitable cationic lipidsfor use in the compositions and methods of the invention include thosedescribed in international patent publication WO 2010/053572,incorporated herein by reference, and most particularly, C12-200described at paragraph [00225] of WO 2010/053572. Another particularlysuitable cationic lipid for use in connection with the invention is2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamineor “DLin-KC2-DMA” (See, WO 2010/042877; Semple et al., nature Biotech.28:172-176 (2010). Other suitable cationic lipids include, for example,5-carboxyspermylglycinedioctadecylamide or “DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.Contemplated cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylammonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,245′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane or “CpLinDMA”,N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-K-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1).

The use of cholesterol-based cationic lipids is also contemplated by thepresent invention. Such cholesterol-based cationic lipids can be used,either alone or in combination with other cationic or non-cationiclipids. Suitable cholesterol-based cationic lipids include, for example,DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335).

In addition, several reagents are commercially available to enhancetransfection efficacy. Suitable examples include LIPOFECTIN (DOTMA:DOPE)(Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen),LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), andEFFECTENE.

Also contemplated are cationic lipids such as the dialkylamino-based,imidazole-based, and guanidinium-based lipids. For example, certainembodiments are directed to a composition comprising one or moreimidazole-based cationic lipids, for example, the imidazole cholesterolester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate. In a preferred embodiment, a carrier(e.g., a lipid nanoparticle) for delivery of RNA (e.g., mRNA) or protein(e.g., an enzyme), for example a therapeutic amount of RNA or protein,may comprise one or more imidazole-based cationic lipids, for example,the imidazole cholesterol ester or “ICE” lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate. The imidazole-based cationic lipids arealso characterized by their reduced toxicity relative to other cationiclipids. The imidazole-based cationic lipids (e.g., ICE) may be used asthe sole cationic lipid in the carrier or lipid nanoparticle, oralternatively may be combined with traditional cationic lipids (e.g.,DOPE, DC-Chol), non-cationic lipids, PEG-modified lipids and/or helperlipids. The cationic lipid may comprise a molar ratio of about 1% toabout 90%, about 2% to about 70%, about 5% to about 50%, about 10% toabout 40% of the total lipid present in the carrier, or preferably about20% to about 70% of the total lipid present in the carrier.

Similarly, certain embodiments are directed to lipid nanoparticlescomprising the HGT4003 cationic lipid2-((2,3-Bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine,as further described in U.S. Provisional Application No. 61/494,882filed Jun. 8, 2011, the entire teachings of which are incorporatedherein by reference in their entirety. In other embodiments thecompositions and methods described herein are directed to lipidnanoparticles comprising one or more ionizable cationic lipids, such as,for example, one or more of the cationic lipids or compounds (e.g.,HGT5001, HGT5002 and HGT5003), as further described in U.S. ProvisionalApplication No. 61/617,468, incorporated herein by reference in theirentirety.

In other embodiments the compositions and methods described herein aredirected to lipid nanoparticles comprising one or more cleavable lipids,such as, for example, one or more cationic lipids or compounds thatcomprise a cleavable disulfide (S—S) functional group (e.g., HGT4001,HGT4002, HGT4003, HGT4004 and HGT4005), as further described in U.S.Provisional Application No. 61/494,882, incorporated herein by referencein their entirety.

The use of polyethylene glycol (PEG)-modified phospholipids andderivatized lipids such as derivatized ceramides (PEG-CER), includingN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000](C8 PEG-2000 ceramide) is also contemplated by the present invention,either alone or preferably in combination with other lipid formulationstogether which comprise the carrier (e.g., a lipid nanoparticle).Contemplated PEG-modified lipids include, but is not limited to, apolyethylene glycol chain of up to 5 kDa in length covalently attachedto a lipid with alkyl chain(s) of C₆-C₂₀ length. The addition of suchcomponents may prevent complex aggregation and may also provide a meansfor increasing circulation lifetime and increasing the delivery of thelipid-polynucleotide composition to the target tissues, (Klibanov et al.(1990) FEBS Letters, 268 (1): 235-237), or they may be selected torapidly exchange out of the formulation in vivo (see U.S. Pat. No.5,885,613). Particularly useful exchangeable lipids are PEG-ceramideshaving shorter acyl chains (e.g., C₁₄ or C₁₈). The PEG-modifiedphospholipid and derivatized lipids of the present invention maycomprise a molar ratio from about 0% to about 20%, about 0.5% to about20%, about 1% to about 15%, about 4% to about 10%, or about 2% of thetotal lipid present in the liposomal carrier. In some embodiments, thePEG-modified lipid employed in the compositions and methods of theinvention is 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol(2000 MW PEG) (DMG-PEG2000).

The present invention also contemplates the use of non-cationic lipidsto facilitate delivery of the nuclease resistant polynucleotides orstabilizing oligonucleotides to one or more target cells, organs ortissues. As used herein, the phrase “non-cationic lipid” refers to anyneutral, zwitterionic or anionic lipid. As used herein, the phrase“anionic lipid” refers to any of a number of lipid species that carry anet negative charge at a selected pH, such as physiological pH.Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. Such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. When used in combination with a cationic lipid, thenon-cationic lipid may comprise a molar ratio of 5% to about 90%, orpreferably about 10% to about 70% of the total lipid present in thecarrier.

Preferably, the carrier (e.g., a lipid nanoparticle) is prepared bycombining multiple lipid and/or polymer components. For example, acarrier may be prepared using DSPC/CHOL/DODAP/C8-PEG-5000 ceramide in amolar ratio of about 1 to 50:5 to 65:5 to 90:1 to 25, respectively. Acarrier may be comprised of additional lipid combinations in variousratios, including for example, DSPC/CHOL/DODAP/mPEG-5000 (e.g., combinedat a molar ratio of about 33:40:25:2), DSPC/CHOL/DODAP/C8 PEG-2000-Cer(e.g., combined at a molar ratio of about 31:40:25:4),POPC/DODAP/C8-PEG-2000-Cer (e.g., combined at a molar ratio of about75-87:3-14:10) or DSPC/CHOL/DOTAP/C8 PEG-2000-Cer (e.g., combined at amolar ratio of about 31:40:25:4). The selection of cationic lipids,non-cationic lipids and/or PEG-modified lipids which comprise theliposomal carrier or lipid nanoparticle, as well as the relative molarratio of such lipids to each other, is based upon the characteristics ofthe selected lipid(s), the nature of the intended target cells ortissues and the characteristics of the polynucleotides to be deliveredby the liposomal carrier. Additional considerations include, forexample, the saturation of the alkyl chain, as well as the size, charge,pH, pKa, fusogenicity and toxicity of the selected lipid(s).

The liposomal carriers for use in the present invention can be preparedby various techniques which are presently known in the art.Multi-lamellar vesicles (MLV) may be prepared by conventionaltechniques, for example, by depositing a selected lipid on the insidewall of a suitable container or vessel by dissolving the lipid in anappropriate solvent, and then evaporating the solvent to leave a thinfilm on the inside of the vessel or by spray drying. An aqueous phasemay then added to the vessel with a vortexing motion which results inthe formation of MLVs. Unilamellar vesicles (ULV) can then be formed byhomogenization, sonication or extrusion of the multi-lamellar vesicles.In addition, unilamellar vesicles can be formed by detergent removaltechniques.

In certain embodiments of this invention, the compositions comprise acarrier wherein a nuclease resistant polynucleotide (e.g., mRNA encodingOTC) is associated on both the surface of the carrier (e.g., a liposome)and encapsulated within the same carrier. For example, duringpreparation of the compositions of the present invention, cationicliposomal carriers may associate with the polynucleotides (e.g., mRNA)through electrostatic interactions with such therapeutic mRNA.

In certain embodiments, the compositions or polynucleotides of thepresent invention may comprise or be loaded with a diagnosticradionuclide, fluorescent material or other material that is detectablein both in vitro and in vivo applications. For example, suitablediagnostic materials for use in the present invention may includeRhodamine-dioleoylphosphatidylethanolamine (Rh-PE), Green FluorescentProtein mRNA (GFP mRNA), Renilla Luciferase mRNA and Firefly LuciferasemRNA.

During the preparation of liposomal carriers, water soluble carrieragents may be encapsulated in the aqueous interior by including them inthe hydrating solution, and lipophilic molecules may be incorporatedinto the lipid bilayer by inclusion in the lipid formulation. In thecase of certain molecules (e.g., cationic or anionic lipophilicpolynucleotides), loading of the polynucleotide into preformed liposomesmay be accomplished, for example, by the methods described in U.S. Pat.No. 4,946,683, the disclosure of which is incorporated herein byreference. Following encapsulation of the polynucleotide, the liposomesmay be processed to remove un-encapsulated mRNA through processes suchas gel chromatography, diafiltration or ultrafiltration. For example, ifit is desirous to remove externally bound polynucleotide from thesurface of the liposomal carrier formulation, such liposomes may besubject to a Diethylaminoethyl SEPHACEL column.

In addition to the encapsulated nuclease resistant polynucleotide, oneor more secondary therapeutic or diagnostic agents may be included inthe carrier. For example, such additional therapeutic agents may beassociated with the surface of the liposome, can be incorporated intothe lipid bilayer of a liposome by inclusion in the lipid formulation orloading into preformed liposomes. (See, e.g., U.S. Pat. Nos. 5,194,654and 5,223,263, which are incorporated by reference herein).

There are several methods for reducing the size, or “sizing”, ofliposomal carriers, and any of these methods may generally be employedwhen sizing is used as part of the invention. The extrusion method is apreferred method of liposome sizing. (Hope, M J et al. Reduction ofLiposome Size and Preparation of Unilamellar Vesicles by ExtrusionTechniques. In: Liposome Technology (G. Gregoriadis, Ed.) Vol. 1. p 123(1993)). The method comprises a step of extruding liposomes through asmall-pore polycarbonate membrane or an asymmetric ceramic membrane toreduce liposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller pore membranes toachieve gradual reduction in liposome size.

A variety of alternative methods known in the art are available forsizing of a population of liposomal carriers. One such sizing method isdescribed in U.S. Pat. No. 4,737,323, the entire teachings of which areincorporated herein by reference. Sonicating a liposome suspensioneither by bath or probe sonication produces a progressive size reductiondown to small ULV less than about 0.05 microns in diameter.Homogenization is another method that relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, MIN are recirculated through a standard emulsion homogenizeruntil selected liposome sizes, typically between about 0.1 and 0.5microns, are observed. The size of the liposomal vesicles may bedetermined by quasi-electric light scattering (QELS) as described inBloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporatedherein by reference. Average liposome diameter may be reduced bysonication of formed liposomes. Intermittent sonication cycles may bealternated with QELS assessment to guide efficient liposome synthesis.

Selection of the appropriate size of a carrier must take intoconsideration the site of the target cell or tissue and to some extentthe application for which the liposome is being made. For example, tothe extent that the compositions are intended for pulmonaryadministration (e.g., as an inhalable liquid or solid carrier), theability of the carrier to distribute into the tissues of the lung may beinfluenced by the size of the carrier particles that comprise suchcomposition. Accordingly, in certain embodiments, it may be desirable toenhance the distribution of such compositions to certain cells ortissues of the lung by appropriately sizing such compositions such thatupon administration (e.g., by inhalation), such compositions distributeto one or more targeted cells and tissues.

In some embodiments, the compositions provided herein are generallyadministered via the pulmonary route of administration. Accordingly, incertain embodiments the carriers and/or compositions disclosed hereinare prepared for pulmonary administration. For example, a pulmonarysurfactant may be added as an excipient component of a carrierformulation (e.g., a lipid nanoparticle comprising one or more cationiclipids, neutral lipids and pulmonary surfactants). Alternatively, incertain embodiments, the compositions disclosed herein may comprise oneor more pulmonary surfactants that may be formulated independently ofthe carrier. The inclusion of pulmonary surfactants (e.g., lamellarbodies) in the compositions disclosed herein may also serve to loosen,break-up or otherwise facilitate the elimination of mucous from thelungs of the subject, thereby improving the distribution of thecompositions into the tissues of the lung. In certain embodiments, suchlamellar bodies may also function as a carrier to facilitate thedelivery or distribution of one or more polynucleotides to target cells,tissues and/or organs. For example, such lamellar body carriers may alsobe loaded or otherwise prepared such that they also comprise one or morepolynucleotides (e.g., mRNA encoding a functional protein or enzyme). Inother embodiments, the compositions disclosed herein may comprisesynthetically- or naturally-prepared lamellar bodies and lipidnanoparticles.

Where the compositions disclosed herein comprise lamellar bodies, suchlamellar bodies may comprise one or more of phosphatidylcholine (PC),phosphatidylethanolamine (PE), phosphatidylserine (PS),phosphatidylinositol (PI), phosphatidylglycerol (PG), sphingomyelin(SM), cholesterol (CHOL) and dipalmitoylphosphatidylcholine (DPPC).

In certain embodiments, the compositions and/or carriers disclosedherein may also comprise one or more exosomes. Exosomes are smallmicro-vesicles that are shed from the surface membranes of most celltypes (e.g., mammalian cell types) and that have been implicated asplaying a pivotal role in cell-to-cell communications (e.g., as avehicle for transferring various bioactive molecules). (See, e.g.,Camussi, et al., Kidney Int. (2010); 78(9): 838-48, the contents ofwhich are incorporated herein by reference in their entirety.)

In certain embodiments, the liver represents an important peripheraltarget organ for the compositions of the present invention in part dueto its central role in metabolism and production of proteins andaccordingly diseases which are caused by defects in liver-specific geneproducts (e.g., the urea cycle disorders) may benefit from specifictargeting of cells (e.g., hepatocytes). Accordingly, in certainembodiments of the present invention, the structural characteristics ofthe target tissue may be exploited to direct the distribution of theliposomal carrier and its polynucleotide payload to such target tissues.For example, to target hepatocytes a liposomal carrier may be sized suchthat its dimensions are smaller than the fenestrations of theendothelial layer lining hepatic sinusoids in the liver; accordingly theliposomal carrier can readily penetrate such endothelial fenestrationsto reach the target hepatocytes. Alternatively, a liposomal carrier maybe sized such that the dimensions of the liposome are of a sufficientdiameter to limit or expressly avoid distribution into certain cells ortissues (e.g., peripheral cells and tissues). For example, a liposomalcarrier may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomal carrier to hepatocytes. Insuch an embodiment, large liposomal carriers will not easily penetratethe endothelial fenestrations, and would instead be cleared by themacrophage Kupffer cells that line the liver sinusoids. Generally, thesize of the carrier is within the range of about 25 to 250 nm,preferably less than about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75nm, 50 nm, 25 nm or 10 nm.

Similarly, the compositions of the present invention may be prepared topreferentially distribute to other local and/or peripheral targettissues, cells or organs, such as the brain, cerebrospinal fluid,muscle, heart, lungs, kidneys and/or spleen. For example, the carriersof the present invention may be prepared to achieve enhanced delivery tothe target cells and tissues. Accordingly, the compositions of thepresent invention may be enriched with additional cationic, non-cationicand PEG-modified lipids to further target tissues or cells.

In certain embodiments, one or more peripheral target cells and tissuesmay function as a biological reservoir or depot capable of expressing orotherwise producing and systemically excreting a functional protein orenzyme, as disclosed for example, in International Application No.PCT/US2010/058457 and in U.S. Provisional Application No. 61/494,881,the teachings of which are both incorporated herein by reference intheir entirety. Accordingly, in certain embodiments of the presentinvention the liposomal carrier may target cells and/or preferentiallydistribute to one or more target cells and tissues (e.g., target cellsand tissues of the liver) following their delivery to a subject.Following transfection of the target cells (e.g., local endothelialcells of the lung), the nuclease resistant mRNA polynucleotides loadedin the carrier are translated and a functional expression productexpressed, excreted and systemically distributed.

In some embodiments, the compositions of the present invention compriseone or more additional molecules (e.g., proteins, peptides, aptamers oroliogonucleotides) which facilitate the transfer of the polynucleotides(e.g., mRNA, miRNA, snRNA and snoRNA) from the carrier into anintracellular compartment of the target cell. In certain embodiments,the additional molecule facilitates the delivery of the polynucleotidesinto, for example, the cytosol, the lysosome, the mitochondrion, thenucleus, the nucleolae or the proteasome of a target cell. Such agentsmay facilitate the transport of the translated protein of interest fromthe cytoplasm to its normal intercellular location (e.g., in themitochondrion) to treat deficiencies in that organelle. In someembodiments, the agent is selected from the group consisting of aprotein, a peptide, an aptamer, and an oligonucleotide. Similarly, incertain embodiments where such agents may exploit the presence of one ormore endogenous receptors or mechanisms to actively transport suchexpressed proteins or enzymes into the plasma. In other embodiments, thecompositions described herein may comprise one or more excipients thatfacilitate the distribution of such compositions into the plasma, wheresuch compositions may further distribute to one or more additionaltarget organs, tissues or cells.

In certain embodiments, the compositions of the present inventionfacilitate a subject's endogenous production of one or more functionalproteins and/or enzymes. The endogenous production or translation ofexogenous nuclease resistant mRNA polynucleotides by a subject toproduce one or more expression products (e.g., proteins and/or enzymes)may, in certain instances demonstrate less immunogenicity relative totheir recombinantly-prepared counterparts that often lack nativepost-translational modifications (e.g., glycosylation). Similarly, theendogenously produced or translated proteins and/or enzymes maydemonstrate more biological activity relative to theirrecombinantly-prepared counterparts. In a preferred embodiment of thepresent invention, the carriers comprise nuclease resistant mRNApolynucleotides which encode a deficient expression product (e.g., aprotein or enzyme). The administration of an mRNA polynucleotide (e.g.,a nuclease resistant mRNA polynucleotide) encoding a deficient proteinor enzyme avoids the need to deliver the polynucleotides to specificorganelles within a target cell (e.g., mitochondria). Rather, upontransfection of a target cell and delivery of the polynucleotides to thecytoplasm of the target cell, the mRNA polynucleotide contents of acarrier may be translated and a functional protein or enzyme expressed.

The present invention also contemplates the discriminatory targeting oftarget cells and tissues by both passive and active targeting means. Thephenomenon of passive targeting exploits the natural distributionspatterns of a carrier in vivo without relying upon the use of additionalexcipients or means to enhance recognition of the carrier by targetcells. For example, carriers which are subject to phagocytosis by thecells of the reticulo-endothelial system are likely to accumulate in theliver or spleen, and accordingly may provide means to passively directthe delivery of the compositions to such target cells.

The present invention also contemplates active targeting, which involvesthe use of additional excipients, referred to herein as “targetingligands” that may be bound (either covalently or non-covalently) to thecarrier to encourage localization of such carrier at certain targetcells or target tissues. For example, targeting may be mediated by theinclusion of one or more endogenous targeting ligands (e.g.,apolipoprotein E) in or on the carrier to encourage distribution to thetarget cells or tissues. Recognition of the targeting ligand by thetarget tissues actively facilitates tissue distribution and cellularuptake of the carrier and/or its polynucleotide contents in the targetcells and tissues (e.g., the inclusion of an apolipoprotein-E targetingligand in or on the carrier may encourage recognition and binding of thecarrier to endogenous low density lipoprotein receptors expressed byhepatocytes). As provided herein, the composition can comprise a ligandcapable of enhancing affinity of the composition to the target cell.Targeting ligands may be linked to the outer bilayer of the lipidparticle during formulation or post-formulation. These methods are wellknown in the art. In addition, some lipid particle formulations mayemploy fusogenic polymers such as PEAA, hemagluttinin, otherlipopeptides (see U.S. patent application Ser. Nos. 08/835,281, and60/083,294, which are incorporated herein by reference) and otherfeatures useful for in vivo and/or intracellular delivery. In other someembodiments, the compositions of the present invention demonstrateimproved transfection efficacies, and/or demonstrate enhancedselectivity towards target cells or tissues of interest. Contemplatedtherefore are compositions which comprise one or more ligands (e.g.,peptides, aptamers, oligonucleotides, a vitamin or other molecules) thatare capable of enhancing the affinity of the compositions and theirpolynucleotide contents for the target cells or tissues. Suitableligands may optionally be bound or linked to the surface of the carrier.In some embodiments, the targeting ligand may span the surface of acarrier or be encapsulated within the carrier. Suitable ligands and areselected based upon their physical, chemical or biological properties(e.g., selective affinity and/or recognition of target cell surfacemarkers or features.) Cell-specific target sites and their correspondingtargeting ligand can vary widely. Suitable targeting ligands areselected such that the unique characteristics of a target cell areexploited, thus allowing the composition to discriminate between targetand non-target cells. For example, compositions of the present inventionmay bear surface markers (e.g., apolipoprotein-B or apolipoprotein-E)that selectively enhance recognition of, or affinity to hepatocytes(e.g., by receptor-mediated recognition of and binding to such surfacemarkers). Additionally, the use of galactose as a targeting ligand wouldbe expected to direct the compositions of the present invention toparenchymal hepatocytes, or alternatively the use of mannose containingsugar residues as a targeting ligand would be expected to direct thecompositions of the present invention to liver endothelial cells (e.g.,mannose containing sugar residues that may bind preferentially to theasialoglycoprotein receptor present in hepatocytes). (See Hillery A M,et al. “Drug Delivery and Targeting: For Pharmacists and PharmaceuticalScientists” (2002) Taylor & Francis, Inc.) The presentation of suchtargeting ligands that have been conjugated to moieties present in thecarrier (e.g., a lipid nanoparticle) therefore facilitate recognitionand uptake of the compositions of the present invention in target cellsand tissues. Examples of suitable targeting ligands include one or morepeptides, proteins, aptamers, vitamins and oligonucleotides.

In certain embodiments, the carriers disclosed herein may also compriseone or more opsonization-inhibiting moieties, which are typically largehydrophilic polymers that are chemically or physically bound to acarrier or vehicle such as a lipid nanoparticle (e.g., by theintercalation of a lipid-soluble anchor into the membrane itself, or bybinding directly to active groups of membrane lipids). Theseopsonization-inhibiting hydrophilic polymers form a protective surfacelayer which significantly decreases the uptake of the pharmaceuticalcarrier or vehicle (e.g., liposomes) by the macrophage-monocyte systemand reticulo-endothelial system, as described for example, in U.S. Pat.No. 4,920,016, the entire disclosure of which is herein incorporated byreference. Carriers modified with opsonization-inhibition moieties thusremain in the circulation much longer than their unmodifiedcounterparts.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, to which the compositions and methods of thepresent invention are administered. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

As used herein, the term “target cell” refers to a cell to which acomposition, nuclease resistant polynucleotide and/or stabilizingoligonucleotide of the invention are to be directed or targeted. In someembodiments, the target cells are deficient in a protein or enzyme ofinterest. In some embodiments, cells are targeted based on their abilityto secrete one or more expression products extracellularly. Thecompositions and methods of the present invention may be prepared topreferentially target a variety of target cells, which include, but arenot limited to, pulmonary epithelial cells (e.g., Type I and IIpneumocytes), alveolar cells, hepatocytes, hematopoietic cells,epithelial cells, endothelial cells, lung cells, bone cells, stem cells,mesenchymal cells, neural cells (e.g., meninges, astrocytes, motorneurons, cells of the dorsal root ganglia and anterior horn motorneurons), photoreceptor cells (e.g., rods and cones), retinal pigmentedepithelial cells, secretory cells, cardiac cells, adipocytes, vascularsmooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells,pituitary cells, synovial lining cells, ovarian cells, testicular cells,fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytesand tumor cells. In certain embodiments, the target cells comprise TypeI pneumocytes, Type II pneumocytes, alveolar cells and combinationsthereof. Following transfection of one or more target cells by thecompositions and nuclease resistant polynucleotides of the presentinvention, expression of the polypeptide, protein or enzyme encoded bysuch polynucleotide may be preferably stimulated and the capability ofsuch target cells to express the protein of interest enhanced. Forexample, transfection of a target cell with a stabilized or duplexedmRNA polynucleotide encoding the OTC enzyme may facilitate the enhancedexpression of the corresponding expression product (OTC) followingtranslation of the mRNA polynucleotide.

Also contemplated by the present inventions are methods of treating asubject having or otherwise affected by a protein or enzyme deficiency.Such methods generally comprise administering to the subject (e.g.,parenterally) a composition comprising a nuclease resistant mRNApolynucleotide and a suitable carrier, wherein the mRNA encodes anenzyme or protein in which the subject is deficient.

The compositions and methods of the present invention may be suitablefor the treatment of diseases or disorders relating to the deficiency ofproteins and/or enzymes. In certain embodiments, the stabilized ornuclease resistant polynucleotides of the present invention encodefunctional proteins or enzymes that are excreted or secreted by thetarget cell into the surrounding extracellular fluid (e.g., mRNAencoding hormones and neurotransmitters). Alternatively, in otherembodiments, the polynucleotides (e.g., mRNA encoding urea cyclemetabolic disorders) of the present invention encode functional proteinsor enzymes that remain in the cytosol of the target cell. Otherdisorders for which the present invention are useful include disorderssuch as Duchenne muscular dystrophy, blood clotting disorders, such ase.g., hemophelia, SMN1-related spinal muscular atrophy (SMA);amyotrophic lateral sclerosis (ALS); GALT-related galactosemia; CysticFibrosis (CF); SLC3A1-related disorders including cystinuria;COL4A5-related disorders including Alport syndrome; galactocerebrosidasedeficiencies; X-linked adrenoleukodystrophy and adrenomyeloneuropathy;Friedreich's ataxia; Pelizaeus-Merzbacher disease; TSC1 and TSC2-relatedtuberous sclerosis; Sanfilippo B syndrome (MPS IIIB); CTNS-relatedcystinosis; the FMR1-related disorders which include Fragile X syndrome,Fragile X-Associated Tremor/Ataxia Syndrome and Fragile X PrematureOvarian Failure Syndrome; Prader-Willi syndrome; hereditary hemorrhagictelangiectasia (AT); Niemann-Pick disease Type Cl; the neuronal ceroidlipofuscinoses-related diseases including Juvenile Neuronal CeroidLipofuscinosis (JNCL), Juvenile Batten disease, Santavuori-Haltiadisease, Jansky-Bielschowsky disease, and PTT-1 and TPP1 deficiencies;EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5-related childhood ataxia withcentral nervous system hypomyelination/vanishing white matter; CACNA1Aand CACNB4-related Episodic Ataxia Type 2; the MECP2-related disordersincluding Classic Rett Syndrome, MECP2-related Severe NeonatalEncephalopathy and PPM-X Syndrome; CDKL5-related Atypical Rett Syndrome;Kennedy's disease (SBMA); Notch-3 related cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL); SCN1A and SCN1B-related seizure disorders; the PolymeraseG-related disorders which include Alpers-Huttenlocher syndrome,POLG-related sensory ataxic neuropathy, dysarthria, andophthalmoparesis, and autosomal dominant and recessive progressiveexternal ophthalmoplegia with mitochondrial DNA deletions; X-Linkedadrenal hypoplasia; X-linked agammaglobulinemia; and Wilson's disease.In certain embodiments, the polynucleotides, and in particular mRNA, ofthe present invention may encode functional proteins or enzymes. Forexample, the compositions of the present invention may include mRNAencoding erythropoietin, α1-antitrypsin, carboxypeptidase N, humangrowth hormone, Factor VII, Factor III, Factor IX, or cystic fibrosistransmembrane conductance regulator (CFTR).

Alternatively the nuclease resistant polynucleotides disclosed hereinmay encode full length antibodies or smaller antibodies both heavy andlight chains) to confer immunity to a subject. While certain embodimentsof the present invention relate to methods and compositions useful forconferring immunity to a subject (e.g., via the translation of mRNApolynucleotides encoding functional antibodies), the inventionsdisclosed herein and contemplated hereby are broadly applicable. In analternative embodiment the compositions of the present invention encodeantibodies that may be used to transiently or chronically affect afunctional response in subjects. For example, the nuclease resistantmRNA polynucleotides of the present invention may encode a functionalmonoclonal or polyclonal antibody, which upon translation (and asapplicable, systemic excretion from the target cells) may be useful fortargeting and/or inactivating a biological target (e.g., a stimulatorycytokine such as tumor necrosis factor). Similarly, the nucleaseresistant mRNA polynucleotides of the present invention may encode, forexample, functional anti-nephritic factor antibodies useful for thetreatment of membranoproliferative glomerulonephritis type II or acutehemolytic uremic syndrome, or alternatively may encode anti-vascularendothelial growth factor (VEGF) antibodies useful for the treatment ofVEGF-mediated diseases, such as cancer.

The compositions of the present invention may be administered and dosedin accordance with current medical practice, taking into account theclinical condition of the subject, the site and method ofadministration, the scheduling of administration, the subject's age,sex, body weight and other factors relevant to clinicians of ordinaryskill in the art. The “effective amount” for the purposes herein may bedetermined by such relevant considerations as are known to those ofordinary skill in experimental clinical research, pharmacological,clinical and medical arts. In some embodiments, the amount administeredis effective to achieve at least some stabilization, improvement orelimination of symptoms and other indicators as are selected asappropriate measures of disease progress, regression or improvement bythose of skill in the art. For example, a suitable amount and dosingregimen is one that causes at least transient expression of the stableor nuclease resistant polynucleotide in the target cell.

Suitable routes of administration of the compositions disclosed hereinmay include, for example, pulmonary, oral, rectal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

In certain embodiments, the compositions of the present invention areformulated such that they are suitable for extended-release of thestabilized or nuclease resistant polynucleotides contained therein. Suchextended-release compositions may be conveniently administered to asubject at extended dosing intervals. For example, in certainembodiments, the compositions of the present invention are administeredto a subject twice day, daily or every other day. In a preferredembodiment, the compositions of the present invention are administeredto a subject twice a week, once a week, every ten days, every two weeks,every three weeks, or more preferably every four weeks, once a month,every six weeks, every eight weeks, every other month, every threemonths, every four months, every six months, every eight months, everynine months or annually. Also contemplated are compositions andliposomal carriers which are formulated for depot administration (e.g.,intramuscularly, subcutaneously, intravitreally) to either deliver orrelease a polynucleotides (e.g., mRNA) over extended periods of time.Preferably, the extended-release means employed are combined withmodifications made to the polynucleotide to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the compounds disclosed herein and relatedmethods for the use of such lyophilized compositions as disclosed forexample, in U.S. Provisional Application No. 61/494,882 filed Jun. 8,2011, the teachings of which are incorporated herein by reference intheir entirety. For example, the lyophilized pharmaceutical compositionsaccording to the invention may be reconstituted prior to theiradministration to a subject (e.g., reconstituted using purified water ornormal saline and inhaled by a subject using a device such as anebulizer). In certain embodiments, the lyophilized compositions can bereconstituted in vivo, for example by lyophilizing such composition inan appropriate dosage form (e.g., an intradermal dosage form such as adisk, rod or membrane) and administering such composition such that itis rehydrated over time in vivo by the individual's bodily fluids.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same. Eachof the publications, reference materials, accession numbers and the likereferenced herein to describe the background of the invention and toprovide additional detail regarding its practice are hereby incorporatedby reference in their entirety.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive teal's,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLES Example 1

The present example illustrates the ability of stabilizingoligonucleotides of the present invention to enhance the production ofprotein when co-administered with non-denatured in vitro transcribedmRNA. Without wishing to be bound by any theory, it is contemplated thatthe stabilizing oligonucleotides modulate the nuclease resistance andincreases the translational efficiency of mRNA polynucleotidetranscripts.

To perform the instant studies, a 15-mer (2′O-Me-uracil) stabilizingoligonucleotide having a phosphorothioate backbone (MW=4965.8 g/mol) wasprepared and which was designed to be complementary to the poly-A tailof an mRNA polynucleotide (MW=299605 g/mol) encoding humanerythropoietin (EPO) protein. The EPO mRNA transcript was contacted withthe stabilizing oligonucleotide at 0.001:1, 0.01:1, 0.1:1, 0.25:1, 1:1,10:1 and 100:1 parts stabilizing oligonucleotide to mRNA polynucleotide.The resultant stabilized mRNA transcripts (designated “0.001”, “0.01”,“0.1”, “0.25”, “1”, “10” or “100”) or the untreated, non-denatured EPOpolynucleotide control transcript (designated “Unhybridized”) were thentransiently transfected into 293T cells. The cumulative amounts of EPOprotein produced and expressed by the transfected 293T cells were thenmeasured at 6, 24 and 72 hour intervals.

As illustrated in FIG. 2 and Table 1, with the exception of thestabilized EPO mRNA transcript prepared using 100:1 parts stabilizingoligonucleotide to mRNA (designated “100”), the cumulative amount of EPOprotein produced and secreted by the 293T cells that were transfectedwith the stabilized mRNA transcripts exceeded the cumulative amount ofEPO protein produced by the cells transfected with the Unhybridized mRNAtranscript. In particular, the stabilized EPO mRNA transcriptsdesignated 0.001, 0.01, 0.1, 0.25, 1 and 10 each resulted in theproduction of more EPO protein relative to the Unhybridized EPO controland, in certain instances exceeded the amount of EPO protein produced bythe control by over 160% at the 6 hour time point.

TABLE 1 Cumulative Amount EPO Produced (%) 6 hr 24 hr 72 hr Unhybridized100 100 100 100 66.5257 57.06707 54.53366 10 153.1145 132.3777 125.67621 161.0291 130.1917 122.7891 0.25 163.0904 139.4457 133.4223 0.1 157.012127.0178 122.2577 0.01 146.0027 121.2735 114.2745 0.001 152.7725150.0675 145.8507 Blank 0 0 0 Lipofectamine 0 0 0

Example 2

The present example further illustrates the ability of the stabilizingoligonucleotides of the present invention to enhance the proteinproduction by first hybridizing to a denatured single-stranded mRNA toform a stabilized mRNA before administering into cells for proteinproduction

As described in Example 1 above, a 15-mer (2′O-Me-uracil) stabilizingoligonucleotide having a phosphorothioate backbone was prepared andwhich was designed to be complementary to the poly-A tail of an mRNApolynucleotide encoding human erythropoietin (EPO) protein. The EPO mRNAtranscript was first denatured at 65° C. for 10 minutes, and thencontacted with the stabilizing oligonucleotide at 0.001:1, 0.01:1,0.1:1, 0.25:1, 1:1, 10:1 and 100:1 parts stabilizing oligonucleotide tomRNA polynucleotide. The resultant stabilized mRNA transcripts(designated “0.001”, “0.01”, “0.1”, “0.25”, “1”, “10” or “100”) or theuntreated, denatured EPO polynucleotide control transcript (designated“Unhybridized”) were then transiently transfected into 293T cells. Thecumulative amounts of EPO protein produced and expressed by thetransfected 293T cells were then measured at 6, 24 and 72 hourintervals.

As illustrated in FIG. 3 and in Table 2 below, relative to the denaturedUnhybridized control mRNA, the percentage of the cumulative amount ofEPO protein produced and secreted by the 293T cells transfected with thestabilized mRNA polynucleotide consistently exceeded the percentage ofthe cumulative amount of EPO protein produced and secreted by theUnhybridized mRNA polynucleotide at each time point evaluated.

TABLE 2 Cumulative Amount EPO Produced (%) 6 hr 24 hr 72 hr Unhybridized100 100 100 100 351.0201 398.7672 383.0498 10 482.7039 586.7506 555.20771 633.1448 685.7419 656.1553 0.25 597.5827 598.5531 572.3968 0.1512.6893 587.4839 554.1234 0.01 697.0948 1062.025 1003.248 0.001281.3981 314.8646 296.2899

For example, the stabilized EPO transcript designated 0.01 demonstratedan approximately 700% increase in the cumulative amount of EPO proteinproduced relative to the Unhybridized control transcript at the 6 hourtime point and in excess of 1,000% at both the 24 hour and 72 hour timepoints. Each of the stabilized mRNA transcripts evaluated werecharacterized by an increase in the cumulative amount of EPO proteinproduced relative to the Unhybridized control.

Example 3

The instant study was performed to investigate optimal length of thestabilizing oligonucleotides of the present invention.

A 30-mer (2′O-Me-uracil) stabilizing oligonucleotide having aphosphorothioate backbone was prepared and which was designed to becomplementary to the poly-A tail of an mRNA polynucleotide encodinghuman erythropoietin (EPO) protein. A non-denatured EPO snRNA transcriptwas contacted with the stabilizing oligonucleotide at 0.001:1, 0.01:1,0.1:1, 0.25:1, 0.5:1, 1:1 and 2:1 parts stabilizing oligonucleotide tomRNA polynucleotide. The resultant stabilized mRNA transcripts(designated “0.001”, “0.01”, “0.1”, “0.25”, “0.5”, “1” or “2”) or theuntreated, non-denatured EPO polynucleotide control transcript(designated “Unhybridized”) were then transiently transfected into 293Tcells. The cumulative amounts of EPO protein produced and expressed bythe transfected 293T cells were then measured at 24, 48, 72 and 96 hourintervals.

As illustrated in FIG. 4 , those stabilized mRNA polynucleotidesprepared using 0.1:1 and 0.5:1 parts stabilizing oligonucleotide to mRNApolynucleotide (designated “0.1” and “0.5”), cumulatively produced andsecreted more EPO protein relative to the Unhybridized controlpolynucleotide. Interestingly, an approximately 10% reduction of thecumulative amount of EPO protein produced relative to the Unhybridizedcontrol polynucleotide was observed with several of the stabilized mRNAtranscripts evaluated (e.g., the stabilized mRNA transcript designated“0.25”). In general, the cumulative amount of EPO protein produced usingthe 30-mer stabilizing oligonucleotide appeared to be less than thatobserved using shorter stabilizing oligonucleotides (e.g., a 15-merstabilizing oligonucleotide). Without wishing to be bound by anyparticular theory, such reduction may be due in part to the greaterdegree of hybridization observed with longer stabilizingoligonucleotides, or the interference with the ability of the mRNAtranscript to form stable secondary structures.

The foregoing examples demonstrate that the stabilized mRNA transcriptsthat were prepared by exposure of the mRNA polynucleotides tostabilizing oligonucleotides produced more protein and demonstratedimproved translational efficiencies relative to those stabilized mRNAtranscripts that were prepared by exposure to the highest ratios ofstabilizing oligonucleotide to mRNA polynucleotide. In particular, thosestabilized mRNA polynucleotides prepared by exposure to about 0.001:1,0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 10:1 parts stabilizingoligonucleotide to mRNA polynucleotide appeared result in more proteinbeing produced and secreted by the transfected cells relative to thenative or un-stabilized mRNA transcript.

Without wishing to be bound by any particular theories, it is believedthat a greater degree of hybridization of the stabilizingoligonucleotide to the mRNA transcript may interfere (e.g., stericallyinterfere) with the ability of the mRNA transcript to form secondarystructures (e.g., hairpin loops) that may serve to further protect andstabilize the mRNA transcript from nuclease degradation. Similarly, agreater degree of hybridization of the mRNA transcript may negativelyimpacting endogenous cellular function, for example, by interfering withthe ability of cells or of organelles within such cells to translate themRNA polynucleotide transcript. The present inventors have also observedthat hybridization of the stabilizing oligonucleotides to the mRNApolynucleotide transcript at lower concentrations (in particular at0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1, 10:1 parts stabilizingoligonucleotide to mRNA polynucleotide) appear to have stabilized themRNA polynucleotide from nuclease degradation, while not materiallyimpacting or negatively interfering with the ability of such stabilizedmRNA transcript to form secondary structures. The exposure of an mRNAtranscript to lower concentrations or ratios of the stabilizingoligonucleotide (e.g., about 0.01:1, 0.1:1, 0.25:1, 0.5:1, 1:1, 2:1,10:1 parts stabilizing oligonucleotide to mRNA polynucleotide) thereforeappears to provide optimum stabilization of mRNA polynucleotidetranscript. Similarly, in certain embodiments, upon hybridizing to anmRNA transcript, the stabilizing oligonucleotides of shorter lengths(e.g., about 15-mer) appear to demonstrate optimal stabilization of themRNA transcript. Accordingly, the foregoing evidences the methods ofmodulating the nuclease resistance of polynucleotides and the improvedtranslational efficiencies observed when polynucleotides are stabilizedwith one or more stabilizing oligonucleotides.

1.-39. (canceled)
 40. A nuclease resistant mRNA comprising mRNA having acoding region and a non-coding region and a complementary stabilizingoligonucleotide hybridized to at least a portion of the non-codingregion of the mRNA, wherein the stabilizing oligonucleotide comprises atleast one modified nucleobase, and wherein the nuclease resistant mRNAis more resistant to nuclease degradation relative to the un-hybridizedmRNA.
 41. (canceled)
 42. (canceled)
 43. The nuclease resistant mRNA ofclaim 40, wherein the nuclease resistant mRNA encodes a protein selectedfrom the group consisting of erythropoietin, human growth hormone,cystic fibrosis transmembrane conductance regulator (CFTR),alpha-galactosidase A, alpha-L-iduronidase, iduronate-2-sulfatase,N-acetylglucosamine-1-phosphate transferase, N-acetylglucosaminidase,alpha-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase,N-acetylgalactosamine-4-sulfatase, beta-glucosidase, galactose-6-sulfatesulfatase, beta-galactosidase, beta-glucuronidase, glucocerebrosidase,heparan sulfamidase, hyaluronidase, galactocerebrosidase, ornithinetranscarbamylase (OTC), carbamoyl-phosphate synthetase 1 (CPS1),argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), andarginase 1 (ARG1).
 44. The nuclease resistant mRNA of claim 40, whereinthe stabilizing oligonucleotide is perfectly complementary to the atleast a portion of the non-coding region of the mRNA.
 45. The nucleaseresistant mRNA of claim 40, wherein the stabilizing oligonucleotide isperfectly complementary to a portion of the coding region of the mRNA.46. The nuclease resistant mRNA of claim 44, wherein the non-codingregion is selected from the group of regions consisting of a 3′untranslated region (UTR), a 5′ untranslated region (UTR), a poly-A tailand a terminal cap.
 47. The nuclease resistant mRNA of claim 40, whereinthe stabilizing oligonucleotide is at least about 1 to about 50nucleotides in length.
 48. The nuclease resistant mRNA of claim 45,wherein the mRNA is an endogenous mRNA of a subject that isinsufficiently expressed by the subject.
 49. (canceled)
 50. The nucleaseresistant mRNA of claim 48, wherein the stabilizing oligonucleotide isadministered to the subject.
 51. (canceled)
 52. The nuclease resistantmRNA of claim 47, wherein the stabilizing oligonucleotide is about 15nucleotides in length.
 53. The nuclease resistant mRNA of claim 40,wherein the stabilizing oligonucleotide comprises two or more modifiednucleobases.
 54. The nuclease resistant mRNA of claim 53, wherein themodified nucleobase comprises a sugar modification.
 55. The nucleaseresistant mRNA of claim 54, wherein the sugar modification is a2′-O-alkyl modification.
 56. The nuclease resistant mRNA of claim 54,wherein the sugar modification comprises at least one modificationselected from the group consisting of a locked polynucleotide (LNA), apeptide polynucleotide (PNA), and combinations thereof.
 57. The nucleaseresistant mRNA of claim 54, wherein the sugar modification is a 2′-sugarmodification selected from the group consisting of a 2′-deoxy-2′-fluoromodification, a 2′-O-methyl modification, a 2′-O-methoxyethylmodification, a 2′-deoxy modification, and combinations thereof.
 58. Thenuclease resistant mRNA of any one of claim 40, wherein the stabilizingoligonucleotide comprises at least one modified internucleoside linkage.59. The nuclease resistant mRNA of claim 58, wherein the modifiedinternucleoside linkage is a phosphorothioate bond.
 60. The nucleaseresistant mRNA of claim 40, wherein the modification comprises at leastone nucleobase modification.
 61. The nuclease resistant mRNA of claim60, wherein the nucleobase modification is selected from the groupconsisting of 5-methyl cytidine, pseudouridine, 2-thio uridine, andcombinations thereof.
 62. The nuclease resistant mRNA of claim 40,wherein the non-coding region comprises a poly(A) tail.
 63. The nucleaseresistant mRNA of claim 62, wherein the stabilizing oligonucleotidecomprises a poly-U sequence. 64.-119. (canceled)